Phenelzine-based probes reveal Secernin-3 is involved in thermal nociception.

Chemical platforms that facilitate both the identification and elucidation of new areas for therapeutic development are necessary but lacking. Activity-based protein profiling (ABPP) leverages active site-directed chemical probes as target discovery tools that resolve activity from expression and immediately marry the targets identified with lead compounds for drug design. However, this approach has traditionally focused on predictable and intrinsic enzyme functionality. Here, we applied our activity-based proteomics discovery platform to map non-encoded and post-translationally acquired enzyme functionalities (e.g. cofactors) in vivo using chemical probes that exploit the nucleophilic hydrazine pharmacophores found in a classic antidepressant drug (e.g. phenelzine, Nardil®). We show the probes are in vivo active and can map proteome-wide tissue-specific target engagement of the drug. In addition to engaging targets (flavoenzymes monoamine oxidase A/B) that are associated with the known therapeutic mechanism as well as several other members of the flavoenzyme family, the probes captured the previously discovered N-terminal glyoxylyl (Glox) group of Secernin-3 (SCRN3) in vivo through a divergent mechanism, indicating this functional feature has biochemical activity in the brain. SCRN3 protein is ubiquitously expressed in the brain, yet gene expression is regulated by inflammatory stimuli. In an inflammatory pain mouse model, behavioral assessment of nociception showed Scrn3 male knockout mice selectively exhibited impaired thermal nociceptive sensitivity. Our study provides a guided workflow to entangle molecular (off)targets and pharmacological mechanisms for therapeutic development.

Phenelzine-based probes reveal Secernin-3 is involved in thermal nociception.

Chemical platforms that facilitate both the identification and elucidation of new areas for therapeutic development are necessary but lacking. Activity-based protein profiling (ABPP) leverages active site-directed chemical probes as target discovery tools that resolve activity from expression and immediately marry the targets identified with lead compounds for drug design. However, this approach has traditionally focused on predictable and intrinsic enzyme functionality. Here, we applied our activity-based proteomics discovery platform to map non-encoded and post-translationally acquired enzyme functionalities (e.g. cofactors) in vivo using chemical probes that exploit the nucleophilic hydrazine pharmacophores found in a classic antidepressant drug (e.g. phenelzine, Nardil ® ). We show the probes are in vivo active and can map proteome-wide tissue-specific target engagement of the drug. In addition to engaging targets (flavoenzymes monoamine oxidase A/B) that are associated with the known therapeutic mechanism as well as several other members of the flavoenzyme family, the probes captured the previously discovered N -terminal glyoxylyl (Glox) group of Secernin-3 (SCRN3) in vivo through a divergent mechanism, indicating this functional feature has biochemical activity in the brain. SCRN3 protein is ubiquitously expressed in the brain, yet gene expression is regulated by inflammatory stimuli. In an inflammatory pain mouse model, behavioral assessment of nociception showed Scrn3 male knockout mice selectively exhibited impaired thermal nociceptive sensitivity. Our study provides a guided workflow to entangle molecular (off)targets and pharmacological mechanisms for therapeutic development.

Cross-Talk and Subset Control of Microglia and Associated Myeloid Cells in Neurological Disorders.

Neurological disorders are highly prevalent and often lead to chronic debilitating disease. Neuroinflammation is a major driver across the spectrum of disorders, and microglia are key mediators of this response, gaining wide acceptance as a druggable cell target. Moreover, clinical providers have limited ability to objectively quantify patient-specific changes in microglia status, which can be a predictor of illness and recovery. This necessitates the development of diagnostic biomarkers and imaging techniques to monitor microglia-mediated neuroinflammation in coordination with neurological outcomes. New insights into the polarization status of microglia have shed light on the regulation of disease progression and helped identify a modifiable target for therapeutics. Thus, the detection and monitoring of microglia activation through the inclusion of diagnostic biomarkers and imaging techniques will provide clinical tools to aid our understanding of the neurologic sequelae and improve long-term clinical care for patients. Recent achievements demonstrated by pre-clinical studies, using novel depletion and cell-targeted approaches as well as single-cell RNAseq, underscore the mechanistic players that coordinate microglial activation status and offer a future avenue for therapeutic intervention.

Neuroblast migration along cellular substrates in the developing porcine brain.

In the past decade it has become evident that neuroblasts continue to supply the human cortex with interneurons via unique migratory streams shortly following birth. Owing to the size of the human brain, these newborn neurons must migrate long distances through complex cellular landscapes to reach their final locations. This process is poorly understood, largely because of technical difficulties in acquiring and studying neurotypical postmortem human samples along with diverging developmental features of well-studied mouse models. We reasoned that migratory streams of neuroblasts utilize cellular substrates, such as blood vessels, to guide their trek from the subventricular zone to distant cortical targets. Here, we evaluate the association between young interneuronal migratory streams and their preferred cellular substrates in gyrencephalic piglets during the developmental equivalent of human birth, infancy, and toddlerhood.

Early Influences of Microbiota on White Matter Development in Germ-Free Piglets.

Abnormalities in the prefrontal cortex (PFC), as well as the underlying white matter (WM) tracts, lie at the intersection of many neurodevelopmental disorders. The influence of microorganisms on brain development has recently been brought into the clinical and research spotlight as alterations in commensal microbiota are implicated in such disorders, including autism spectrum disorders, schizophrenia, depression, and anxiety via the gut-brain axis. In addition, gut dysbiosis is common in preterm birth patients who often display diffuse WM injury and delayed WM maturation in critical tracts including those within the PFC and corpus callosum. Microbial colonization of the gut aligns with ongoing postnatal processes of oligodendrogenesis and the peak of brain myelination in humans; however, the influence of microbiota on gyral WM development remains elusive. Here, we develop and validate a neonatal germ-free swine model to address these issues, as piglets share key similarities in WM volume, developmental trajectories, and distribution to humans. We find significant region-specific reductions, and sexually dimorphic trends, in WM volume, oligodendrogenesis, and mature oligodendrocyte numbers in germ-free piglets during a key postnatal epoch of myelination. Our findings indicate that microbiota plays a critical role in promoting WM development during early life when the brain is vulnerable to environmental insults that can result in an array of disabilities manifesting later in life.

Clearing techniques for visualizing the nervous system in development, injury, and disease.

Modern clearing techniques enable high resolution visualization and 3D reconstruction of cell populations and their structural details throughout large biological samples, including intact organs and even entire organisms. In the past decade, these methods have become more tractable and are now being utilized to provide unforeseen insights into the complexities of the nervous system. While several iterations of optical clearing techniques have been developed, some are more suitable for specific applications than others depending on the type of specimen under study. Here we review findings from select studies utilizing clearing methods to visualize the developing, injured, and diseased nervous system within numerous model systems and species. We note trends and imbalances in the types of research questions being addressed with clearing methods across these fields in neuroscience. In addition, we discuss restrictions in applying optical clearing methods for postmortem tissue from humans and large animals and emphasize the lack in continuity between studies of these species. We aim for this review to serve as a key outline of available tissue clearing methods used successfully to address issues across neuronal development, injury/repair, and aging/disease.

PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis.

PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy. PINK1 and Parkin have been proposed to act as tumor suppressors, as loss-of-function mutations are correlated with enhanced tumorigenesis. However, it is unclear how PINK1 and Parkin act in coordination during mitophagy to influence the cell cycle. Here we show that PINK1 and Parkin genetically interact with proteins involved in cell cycle regulation, and loss of PINK1 and Parkin accelerates cell growth. PINK1- and Parkin-mediated activation of TBK1 at the mitochondria during mitophagy leads to a block in mitosis due to the sequestration of TBK1 from its physiological role at centrosomes during mitosis. Our study supports a diverse role for the far-reaching, regulatory effects of mitochondrial quality control in cellular homeostasis and demonstrates that the PINK1/Parkin pathway genetically interacts with the cell cycle, providing a framework for understanding the molecular basis linking PINK1 and Parkin to mitosis.

Treatment With Tetrahydrobiopterin Improves White Matter Maturation in a Mouse Model for Prenatal Hypoxia in Congenital Heart Disease.

Background Reduced oxygen delivery in congenital heart disease causes delayed brain maturation and white matter abnormalities in utero. No treatment currently exists. Tetrahydrobiopterin (BH4) is a cofactor for neuronal nitric oxide synthase. BH4 availability is reduced upon NOS activation, such as during hypoxic conditions, and leads to toxin production. We hypothesize that BH4 levels are depleted in the hypoxic brain and that BH4 replacement therapy mitigates the toxic effects of hypoxia on white matter. Methods and Results Transgenic mice were used to visualize oligodendrocytes. Hypoxia was introduced during a period of white matter development equivalent to the human third trimester. BH4 was administered during hypoxia. BH4 levels were depleted in the hypoxic brain by direct quantification (n=7-12). The proliferation (n=3-6), apoptosis (n=3-6), and developmental stage (n=5-8) of oligodendrocytes were determined immunohistologically. Total oligodendrocytes increased after hypoxia, consistent with hypoxia-induced proliferation seen previously; however, mature oligodendrocytes were less prevalent in hypoxia, and there was accumulation of immature oligodendrocytes. BH4 treatment improved the mature oligodendrocyte number such that it did not differ from normoxia, and accumulation of immature oligodendrocytes was not observed. These results persisted beyond the initial period of hypoxia (n=3-4). Apoptosis increased with hypoxia but decreased with BH4 treatment to normoxic levels. White matter myelin levels decreased following hypoxia by western blot. BH4 treatment normalized myelination (n=6-10). Hypoxia worsened sensory-motor coordination on balance beam tasks, and BH4 therapy normalized performance (n=5-9). Conclusions Suboptimal BH4 levels influence hypoxic white matter abnormalities. Repurposing BH4 for use during fetal brain development may limit white matter dysmaturation in congenital heart disease.

Angiopoietin/Tie2 Axis Regulates the Age-at-Injury Cerebrovascular Response to Traumatic Brain Injury.

Although age-at-injury influences chronic recovery from traumatic brain injury (TBI), the differential effects of age on early outcome remain understudied. Using a male murine model of moderate contusion injury, we investigated the underlying mechanism(s) regulating the distinct response between juvenile and adult TBI. We demonstrate similar biomechanical and physical properties of naive juvenile and adult brains. However, following controlled cortical impact (CCI), juvenile mice displayed reduced cortical lesion formation, cell death, and behavioral deficits at 4 and 14 d. Analysis of high-resolution laser Doppler imaging showed a similar loss of cerebral blood flow (CBF) in the ipsilateral cortex at 3 and 24 h post-CCI, whereas juvenile mice showed enhanced subsequent restoration at 2-4 d compared with adults. These findings correlated with reduced blood-brain barrier (BBB) disruption and increased perilesional vessel density. To address whether an age-dependent endothelial cell (EC) response affects vessel stability and tissue outcome, we magnetically isolated CD31+ ECs from sham and injured cortices and evaluated mRNA expression. Interestingly, we found increased transcripts for BBB stability-related genes and reduced expression of BBB-disrupting genes in juveniles compared with adults. These differences were concomitant with significant changes in miRNA-21-5p and miR-148a levels. Accompanying these findings was robust GFAP immunoreactivity, which was not resolved by day 35. Importantly, pharmacological inhibition of EC-specific Tie2 signaling abolished the juvenile protective effects. These findings shed new mechanistic light on the divergent effects that age plays on acute TBI outcome that are both spatial and temporal dependent.SIGNIFICANCE STATEMENT Although a clear "window of susceptibility" exists in the developing brain that could deter typical developmental trajectories if exposed to trauma, a number of preclinical models have demonstrated evidence of early recovery in younger patients. Our findings further demonstrate acute neuroprotection and improved restoration of cerebral blood flow in juvenile mice subjected to cortical contusion injury compared with adults. We also demonstrate a novel role for endothelial cell-specific Tie2 signaling in this age-related response, which is known to promote barrier stability, is heightened in the injured juvenile vasculature, and may be exploited for therapeutic interventions across the age spectrum following traumatic brain injury.

An Implantable Micro-Caged Device for Direct Local Delivery of Agents.

Local and controlled delivery of therapeutic agents directly into focally afflicted tissues is the ideal for the treatment of diseases that require direct interventions. However, current options are obtrusive, difficult to implement, and limited in their scope of utilization; the optimal solution requires a method that may be optimized for available therapies and is designed for exact delivery. To address these needs, we propose the Biocage, a customizable implantable local drug delivery platform. The device is a needle-sized porous container capable of encasing therapeutic molecules and matrices of interest to be eluted into the region of interest over time. The Biocage was fabricated using the Nanoscribe Photonic Professional GT 3D laser lithography system, a two-photon polymerization (2PP) 3D printer capable of micron-level precision on a millimeter scale. We demonstrate the build consistency and features of the fabricated device; its ability to release molecules; and a method for its accurate, stable delivery in mouse brain tissue. The Biocage provides a powerful tool for customizable and precise delivery of therapeutic agents into target tissues.

Microstructural Alterations and Oligodendrocyte Dysmaturation in White Matter After Cardiopulmonary Bypass in a Juvenile Porcine Model.

BACKGROUND: Newly developed white matter (WM) injury is common after cardiopulmonary bypass (CPB) in severe/complex congenital heart disease. Fractional anisotropy (FA) allows measurement of macroscopic organization of WM pathology but has rarely been applied after CPB. The aims of our animal study were to define CPB-induced FA alterations and to determine correlations between these changes and cellular events after congenital heart disease surgery. METHODS AND RESULTS: Normal porcine WM development was first assessed between 3 and 7 weeks of age: 3-week-old piglets were randomly assigned to 1 of 3 CPB-induced insults. FA was analyzed in 31 WM structures. WM oligodendrocytes, astrocytes, and microglia were assessed immunohistologically. Normal porcine WM development resembles human WM development in early infancy. We found region-specific WM vulnerability to insults associated with CPB. FA changes after CPB were also insult dependent. Within various WM areas, WM within the frontal cortex was susceptible, suggesting that FA in the frontal cortex should be a biomarker for WM injury after CPB. FA increases occur parallel to cellular processes of WM maturation during normal development; however, they are altered following surgery. CPB-induced oligodendrocyte dysmaturation, astrogliosis, and microglial expansion affect these changes. FA enabled capturing CPB-induced cellular events 4 weeks postoperatively. Regions most resilient to CPB-induced FA reduction were those that maintained mature oligodendrocytes. CONCLUSIONS: Reducing alterations of oligodendrocyte development in the frontal cortex can be both a metric and a goal to improve neurodevelopmental impairment in the congenital heart disease population. Studies using this model can provide important data needed to better interpret human imaging studies.

Neurodevelopmental Abnormalities and Congenital Heart Disease: Insights Into Altered Brain Maturation.

In the past 2 decades, it has become evident that individuals born with congenital heart disease (CHD) are at risk of developing life-long neurological deficits. Multifactorial risk factors contributing to neurodevelopmental abnormalities associated with CHD have been identified; however, the underlying causes remain largely unknown, and efforts to address this issue have only recently begun. There has been a dramatic shift in focus from newly acquired brain injuries associated with corrective and palliative heart surgery to antenatal and preoperative factors governing altered brain maturation in CHD. In this review, we describe key time windows of development during which the immature brain is vulnerable to injury. Special emphasis is placed on the dynamic nature of cellular events and how CHD may adversely impact the cellular units and networks necessary for proper cognitive and motor function. In addition, we describe current gaps in knowledge and offer perspectives about what can be done to improve our understanding of neurological deficits in CHD. Ultimately, a multidisciplinary approach will be essential to prevent or improve adverse neurodevelopmental outcomes in individuals surviving CHD.

Abnormal neurogenesis and cortical growth in congenital heart disease.

Long-term neurological deficits due to immature cortical development are emerging as a major challenge in congenital heart disease (CHD). However, cellular mechanisms underlying dysregulation of perinatal corticogenesis in CHD remain elusive. The subventricular zone (SVZ) represents the largest postnatal niche of neural stem/progenitor cells (NSPCs). We show that the piglet SVZ resembles its human counterpart and displays robust postnatal neurogenesis. We present evidence that SVZ NSPCs migrate to the frontal cortex and differentiate into interneurons in a region-specific manner. Hypoxic exposure of the gyrencephalic piglet brain recapitulates CHD-induced impaired cortical development. Hypoxia reduces proliferation and neurogenesis in the SVZ, which is accompanied by reduced cortical growth. We demonstrate a similar reduction in neuroblasts within the SVZ of human infants born with CHD. Our findings demonstrate that SVZ NSPCs contribute to perinatal corticogenesis and suggest that restoration of SVZ NSPCs' neurogenic potential is a candidate therapeutic target for improving cortical growth in CHD.

Transient mitochondrial DNA double strand breaks in mice cause accelerated aging phenotypes in a ROS-dependent but p53/p21-independent manner.

We observed that the transient induction of mtDNA double strand breaks (DSBs) in cultured cells led to activation of cell cycle arrest proteins (p21/p53 pathway) and decreased cell growth, mediated through reactive oxygen species (ROS). To investigate this process in vivo we developed a mouse model where we could transiently induce mtDNA DSBs ubiquitously. This transient mtDNA damage in mice caused an accelerated aging phenotype, preferentially affecting proliferating tissues. One of the earliest phenotypes was accelerated thymus shrinkage by apoptosis and differentiation into adipose tissue, mimicking age-related thymic involution. This phenotype was accompanied by increased ROS and activation of cell cycle arrest proteins. Treatment with antioxidants improved the phenotype but the knocking out of p21 or p53 did not. Our results demonstrate that transient mtDNA DSBs can accelerate aging of certain tissues by increasing ROS. Surprisingly, this mtDNA DSB-associated senescence phenotype does not require p21/p53, even if this pathway is activated in the process.

Hypoxia diminishes the protective function of white-matter astrocytes in the developing brain.

OBJECTIVES: White-matter injury after surgery is common in neonates with cerebral immaturity secondary to in utero hypoxia. Astrocytes play a central role in brain protection; however, the reaction of astrocytes to hypothermic circulatory arrest (HCA) remains unknown. We investigated the role of astrocytes in white-matter injury after HCA and determined the effects of preoperative hypoxia on this role, using a novel mouse model. METHODS: Mice were exposed to hypoxia from days 3 to 11, which is equivalent to the third trimester in humans (prehypoxia, n = 49). Brain slices were transferred to a chamber perfused by cerebrospinal fluid. Oxygen-glucose deprivation (OGD) was performed to simulate ischemia-reperfusion/reoxygenation resulting from circulatory arrest under hypothermia. Astrocyte reactions were compared with preoperative normoxia (prenormoxia; n = 45). RESULTS: We observed astrocyte activation after 25°C ischemia-reperfusion/reoxygenation in prenormoxia (P < .01). Astrocyte number after OGD correlated with caspase-3(+) cells (rho = .77, P = .001), confirming that astrogliosis is an important response after HCA. At 3 hours after OGD, astrocytes in prenormoxia had already proliferated and become activated (P < .05). Conversely, astrocytes that developed under hypoxia did not display these responses. At 20 hours after ischemia-reperfusion/reoxygenation, astrogliosis was not observed in prehypoxia, demonstrating that hypoxia altered the response of astrocytes to insult. In contrast to prenormoxia, caspase-3(+) cells in prehypoxia increased after ischemia reperfusion/reoxygenation, compared with control (P < .01). Caspase-3(+) cells were more common with prehypoxia than with prenormoxia (P < .001), suggesting that lack of astrogliosis permits increased white-matter injury. CONCLUSIONS: Preoperative hypoxia alters the neuroprotective function of astrocytes. Restoring this function before surgery may be a therapeutic option to reduce postoperative white-matter injury in the immature brain.

Endothelin-B Receptor Activation in Astrocytes Regulates the Rate of Oligodendrocyte Regeneration during Remyelination.

Reactive astrogliosis is an essential and ubiquitous response to CNS injury, but in some cases, aberrant activation of astrocytes and their release of inhibitory signaling molecules can impair endogenous neural repair processes. Our lab previously identified a secreted intercellular signaling molecule, called endothelin-1 (ET-1), which is expressed at high levels by reactive astrocytes in multiple sclerosis (MS) lesions and limits repair by delaying oligodendrocyte progenitor cell (OPC) maturation. However, as ET receptors are widely expressed on neural cells, the cell- and receptor-specific mechanisms of OPC inhibition by ET-1 action remain undefined. Using pharmacological approaches and cell-specific endothelin receptor (EDNR) ablation, we show that ET-1 acts selectively through EDNRB on astrocytes--and not OPCs--to indirectly inhibit remyelination. These results demonstrate that targeting specific pathways in reactive astrocytes represents a promising therapeutic target in diseases with extensive reactive astrogliosis, including MS.

Prolonged White Matter Inflammation After Cardiopulmonary Bypass and Circulatory Arrest in a Juvenile Porcine Model.

BACKGROUND: White matter (WM) injury is common after neonatal cardiopulmonary bypass (CPB). We have demonstrated that the inflammatory response to CPB is an important mechanism of WM injury. Microglia are brain-specific immune cells that respond to inflammation and can exacerbate injury. We hypothesized that microglia activation contributes to WM injury caused by CPB. METHODS: Juvenile piglets were randomly assigned to 1 of 3 CPB-induced brain insults (1, no-CPB; 2, full-flow CPB; 3, CPB and circulatory arrest). Neurobehavioral tests were performed. Animals were sacrificed 3 days or 4 weeks postoperatively. Microglia and proliferating cells were immunohistologically identified. Seven analyzed WM regions were further categorized into 3 fiber connections (1, commissural; 2, projection; 3, association fibers). RESULTS: Microglia numbers significantly increased on day 3 after CPB and circulatory arrest, but not after full-flow CPB. Fiber categories did not affect these changes. On post-CPB week 4, proliferating cell number, blood leukocyte number, interleukin (IL)-6 levels, and neurologic scores had normalized. However, both full-flow CPB and CPB and circulatory arrest displayed significant increases in the microglia number compared with control. Thus brain-specific inflammation after CPB persists despite no changes in systemic biomarkers. Microglia number was significantly different among fiber categories, being highest in association and lowest in commissural connections. Thus there was a WM fiber-dependent microglia reaction to CPB. CONCLUSIONS: This study demonstrates prolonged microglia activation in WM after CPB. This brain-specific inflammatory response is systemically silent. It is connection fiber-dependent which may impact specific connectivity deficits observed after CPB. Controlling microglia activation after CPB is a potential therapeutic intervention to limit neurologic deficits after CPB.

Congenital cardiac anomalies and white matter injury.

Cardiac abnormalities are the most common birth defects. Derangement of circulatory flow affects many vital organs; without proper supply of oxygenated blood, the brain is particularly vulnerable. Although surgical interventions have greatly reduced mortality rates, patients often suffer an array of neurological deficits throughout life. Neuroimaging provides a macroscopic assessment of brain injury and has shown that white matter (WM) is at risk. Oligodendrocytes and myelinated axons have been identified as major targets of WM injury, but still little is known about how congenital heart anomalies affect the brain at the cellular level. Further integration of animal model studies and clinical research will define novel therapeutic targets and new standards of care to prevent developmental delay associated with cardiac abnormalities.

Astrocytes play a key role in EAE pathophysiology by orchestrating in the CNS the inflammatory response of resident and peripheral immune cells and by suppressing remyelination.

Astrocytes respond to insult with a process of cellular activation known as reactive astrogliosis. One of the key signals regulating this phenomenon is the transcription factor nuclear factor-kappa B (NF-κB), which is responsible for modulating inflammation, cell survival, and cell death. In astrocytes, following trauma or disease, the expression of NF-κB-dependent genes is highly activated. We previously demonstrated that inactivation of astroglial NF-κB in vivo (GFAP-IκBα-dn mice) leads to improved functional outcome in experimental autoimmune encephalomyelitis (EAE), and this is accompanied by reduction of pro-inflammatory gene expression in the CNS. Here we extend our studies to show that recovery from EAE in GFAP-IκBα-dn mice is associated with reduction of peripheral immune cell infiltration into the CNS at the chronic phase of EAE. This is not dependent on a less permeable blood-brain barrier, but rather on a reduced immune cell mobilization from the periphery. Furthermore, once inside the CNS, the ability of T cells to produce pro-inflammatory cytokines is diminished during acute disease. In parallel, we found that the number of total and activated microglial cells is reduced, suggesting that functional improvement in GFAP-IκBα-dn mice is dependent upon reduction of the overall inflammatory response within the CNS sustained by both resident and infiltrating cells. This results in preservation of myelin compaction and enhanced remyelination, as shown by electron microscopy analysis of the spinal cord. Collectively our data indicate that astrocytes are key players in driving CNS inflammation and are directly implicated in the pathophysiology of EAE, since blocking their pro-inflammatory capability results in protection from the disease.

Inhibition of NADPH oxidase activation in oligodendrocytes reduces cytotoxicity following trauma.

Spinal cord injury is a debilitating neurological disorder that initiates a cascade of cellular events that result in a period of secondary damage that can last for months after the initial trauma. The ensuing outcome of these prolonged cellular perturbations is the induction of neuronal and glial cell death through excitotoxic mechanisms and subsequent free radical production. We have previously shown that astrocytes can directly induce oligodendrocyte death following trauma, but the mechanisms regulating this process within the oligodendrocyte remain unclear. Here we provide evidence demonstrating that astrocytes directly regulate oligodendrocyte death after trauma by inducing activation of NADPH oxidase within oligodendrocytes. Spinal cord injury resulted in a significant increase in oxidative damage which correlated with elevated expression of the gp91 phox subunit of the NADPH oxidase enzyme. Immunohistochemical analysis confirmed the presence of gp91 phox in oligodendrocytes in vitro and at 1 week following spinal cord injury. Exposure of oligodendrocytes to media from injured astrocytes resulted in an increase in oligodendrocyte NADPH oxidase activity. Inhibition of NADPH oxidase activation was sufficient to attenuate oligodendrocyte death in vitro and at 1 week following spinal cord injury, suggesting that excitotoxicity of oligodendrocytes after trauma is dependent on the intrinsic activation of the NADPH oxidase enzyme. Acute administration of the NADPH oxidase inhibitor apocynin and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate channel blocker 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione significantly improved locomotor behavior and preserved descending axon fibers following spinal cord injury. These studies lead to a better understanding of oligodendrocyte death after trauma and identify potential therapeutic targets in disorders involving demyelination and oligodendrocyte death.