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Neuroactive steroids reduce mortality, decrease edema, and improve functional outcomes in preclinical and clinical traumatic brain injury (TBI) studies. In this study, we tested the efficacy of two related novel neuroactive steroids, NTS-104 and NTS-105, in a rat model of TBI. NTS-104 is a water-soluble prodrug of NTS-105, a partial progesterone receptor agonist. To investigate the effects of NTS-104 on TBI recovery, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion injury or sham surgery and were treated with vehicle or NTS-104 (10 ​mg/kg, intramuscularly) at 4, 10, 24, and 48 ​h post-TBI. The therapeutic time window was also assessed using the neuroactive steroid NTS-105 (3 ​mg/kg, intramuscularly). Edema in the parietal cortex and hippocampus, measured at 3 days post-injury (DPI), was reduced by NTS-104 and NTS-105. NTS-105 was effective in reducing edema when given at 4, 10, or 24 ​h post-injury. Sensorimotor deficits in the cylinder test at 3 DPI were ameliorated by NTS-104 and NTS-105 treatment. Cognitive recovery, assessed with cue and contextual fear conditioning and retention of the water maze task assessed subacutely 1-3 weeks post-injury, also improved with NTS-104 treatment. Cortical and hippocampal atrophy at 22 DPI did not improve, indicating that NTS-104/NTS-105 may promote post-TBI cognitive recovery by controlling edema and other processes. These results demonstrate that NTS-104/NTS-105 is a promising therapeutic approach to improve motor and cognitive recovery after moderate TBI., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Traumatic brain injury (TBI) leads to changes in the neural circuitry of the hippocampus that result in chronic learning and memory deficits. However, effective therapeutic strategies to ameliorate these chronic learning and memory impairments after TBI are limited. Two pharmacological targets for enhancing cognition are nicotinic acetylcholine receptors (nAChRs) and GABA A receptors (GABA A Rs), both of which regulate hippocampal network activity to form declarative memories. A promising compound, 522-054, both allosterically enhances α7 nAChRs and inhibits α5 subunit-containing GABA A Rs. Administration of 522-054 enhances long-term potentiation (LTP) and cognitive functioning in non-injured animals. In this study, we assessed the effects of 522-054 on hippocampal synaptic plasticity and learning and memory deficits in the chronic post-TBI recovery period. Adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury or sham surgery. At 12 wk after injury, we assessed basal synaptic transmission and LTP at the Schaffer collateral-CA1 synapse of the hippocampus. Bath application of 522-054 to hippocampal slices reduced deficits in basal synaptic transmission and recovered TBI-induced impairments in LTP. Moreover, treatment of animals with 522-054 at 12 wk post-TBI improved cue and contextual fear memory and water maze acquisition and retention without a measurable effect on cortical or hippocampal atrophy. These results suggest that dual allosteric modulation of α7 nAChR and α5 GABA A R signaling may be a potential therapy for treating cognitive deficits during chronic recovery from TBI., Competing Interests: Declaration of competing interest Balleste, Sangadi, Titus, and Atkins have no competing interests to declare. Gee, Johnstone, and Hogenkamp are patent authors on US Pat. 8,865,719 which covers composition of matter and method of use claims associated with 522–054., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cognitive losses resulting from severe brain trauma have long been associated with the focal region of tissue damage, leading to devastating functional impairment. For decades, researchers have focused on the sequelae of cellular alterations that exist within the perilesional tissues; however, few clinical trials have been successful. Here, we employed a mouse brain injury model that resulted in expansive synaptic damage to regions outside the focal injury. Our findings demonstrate that synaptic damage results from the prolonged increase in D-serine release from activated microglia and astrocytes, which leads to hyperactivation of perisynaptic NMDARs, tagging of damaged synapses by complement components, and the reactivation of developmental pruning processes. We show that this mechanistic pathway is reversible at several stages within a prolonged and progressive period of synaptic loss. Importantly, these key factors are present in acutely injured brain tissue acquired from patients with brain injury, which supports a therapeutic neuroprotective strategy.

Traumatic brain injury (TBI) results in several pathological changes within the hippocampus that result in adverse effects on learning and memory. Therapeutic strategies to enhance learning and memory after TBI are still in the early stages of clinical development. One strategy is to target the α7 nicotinic acetylcholine receptor (nAChR), which is highly expressed in the hippocampus and contributes to the formation of long-term memory. In our previous study, we found that AVL-3288, a positive allosteric modulator of the α7 nAChR, improved cognitive recovery in rats after moderate fluid-percussion injury (FPI). However, whether AVL-3288 improved cognitive recovery specifically through the α7 nAChR was not definitively determined. In this study we utilized Chrna7 knockout mice and compared their recovery to wild-type mice treated with AVL-3288 after TBI. We hypothesized that AVL-3288 treatment would improve learning and memory in wild-type mice, but not Chrna7 -/- mice after TBI. Adult male C57BL/6 wild-type and Chrna7 -/- mice received sham surgery or moderate controlled cortical impact (CCI) and recovered for 3 months. Mice were then treated with vehicle or AVL-3288 at 30 min prior to contextual fear conditioning. At 3 months after CCI, expression of α7 nAChR, choline acetyltransferase (ChAT), high-affinity choline transporter (ChT), and vesicular acetylcholine transporter (VAChT) were found to be significantly decreased in the hippocampus. Treatment of wild-type mice at 3 months after CCI with AVL-3288 significantly improved cue and contextual fear conditioning, whereas no beneficial effects were observed in Chrna7 -/- mice. Parietal cortex and hippocampal atrophy were not improved with AVL-3288 treatment in either wild-type or Chrna7 -/- mice. Our results indicate that AVL-3288 improves cognition during the chronic recovery phase of TBI through modulation of the α7 nAChR., Competing Interests: Declaration of Competing Interest Gee, Johnstone, and Hogenkamp are patent authors on US Pat. 7,820,663 which covers composition of matter and method of use claims associated with AVL-3288., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In recent years, significant advances have been made in the study of mild traumatic brain injury (mTBI). Complete recovery from mTBI normally requires days to weeks, yet a subset of the population suffers from symptoms for weeks to months after injury. The risk factors for these prolonged symptoms have not yet been fully understood. In this chapter, we address one proposed risk factor, early life stress (ELS) and its influence on mTBI recovery. To study the effects of ELS on mTBI recovery, accepted animal models of ELS, including maternal separation, limited bedding and nesting, and chronic unpredictable stress, have been implemented. Combining these ELS models with standardized mTBI models, such as fluid percussion injury or controlled cortical impact, has allowed for a deeper understanding of the neuronal, hormonal, and cognitive changes that occur after mTBI following ELS. These preclinical findings are being used to understand how adverse childhood experiences may predispose a subset of individuals to poorer recovery after mTBI., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in communication, and social skills, as well as repetitive and/or restrictive interests and behaviors. The severity of ASD varies from mild to severe, drastically interfering with the quality of life of affected individuals. The current occurrence of ASD in the United States is about 1 in 44 children. The precise pathophysiology of ASD is still unknown, but it is believed that ASD is heterogeneous and can arise due to genetic etiology. Although various genes have been implicated in predisposition to ASD, metabotropic glutamate receptor 5 (mGluR5) is one of the most common downstream targets, which may be involved in autism. mGluR5 signaling has been shown to play a crucial role in neurodevelopment and neural transmission making it a very attractive target for understanding the pathogenesis of ASD. In the present study, we determined the effect of genetic ablation of mGluR5 (Grm5) on an ASD-like phenotype using a rat model to better understand the role of mGluR5 signaling in behavior patterns and clinical manifestations of ASD. We observed that mGluR5 Ko rats exhibited exaggerated self-grooming and increased marble burying, as well as deficits in social novelty. Our results suggest that mGluR5 Ko rats demonstrate an ASD-like phenotype, specifically impaired social interaction as well as repetitive and anxiety-like behavior, which are correlates of behavior symptoms observed in individuals with ASD. The mGluR5 Ko rat model characterized in this study may be explored to understand the molecular mechanisms underlying ASD and for developing effective therapeutic modalities., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Eshraghi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The neurocognitive impairments associated with mild traumatic brain injury (TBI) often resolve within 1-2 weeks; however, a subset of people exhibit persistent cognitive dysfunction for weeks to months after injury. The factors that contribute to these persistent deficits are unknown. One potential risk factor for worsened outcome after TBI is a history of stress experienced by a person early in life. Early life stress (ELS) includes maltreatment such as neglect, and interferes with the normal construction of cortical and hippocampal circuits. We hypothesized that a history of ELS contributes to persistent learning and memory dysfunction following a TBI. To explore this interaction, we modeled ELS by separating Sprague Dawley pups from their nursing mothers from post-natal days 2-14 for 3 h daily. At 2 months of age, male rats received sham surgery or mild to moderate parasagittal fluid-percussion brain injury. We found that the combination of ELS with TBI in adulthood impaired hippocampal-dependent learning, as assessed with contextual fear conditioning, the water maze task, and spatial working memory. Cortical atrophy was significantly exacerbated in TBI animals exposed to ELS compared with normal-reared TBI animals. Changes in corticosterone in response to restraint stress were prolonged in TBI animals that received ELS compared with TBI animals that were normally reared or sham animals that received ELS. Our findings indicate that ELS is a risk factor for worsened outcome after TBI, and results in persistent learning and memory deficits, worsened cortical pathology, and an exacerbation of the hormonal stress response.

Background and Purpose: A significant number of HIV-1 patients on antiretroviral therapy develop HIV-associated neurocognitive disorders (HAND). Evidence indicate that biological sex may regulate HAND pathogenesis, but the mechanisms remain unknown. We investigated synaptic mechanisms associated with sex differences in HAND, using the HIV-1-transgenic 26 (Tg26) mouse model., Experimental Approach: Contextual- and cue-dependent memories of male and female Tg26 mice and littermate wild type mice were assessed in a fear conditioning paradigm. Hippocampal electrophysiology, immunohistochemistry, western blot, qRT-PCR and ELISA techniques were used to investigate cellular, synaptic and molecular impairments., Key Results: Cue-dependent memory was unaltered in male and female Tg26 mice, when compared to wild type mice. Male, but not female, Tg26 mice showed deficits in contextual fear memory. Consistently, only male Tg26 mice showed depressed hippocampal basal synaptic transmission and impaired LTP induction in area CA1. These deficits in male Tg26 mice were independent of hippocampal neuronal loss and microglial activation but were associated with increased HIV-1 long terminal repeat mRNA expression, reduced hippocampal synapsin-1 protein, reduced BDNF mRNA and protein, reduced AMPA glutamate receptor (GluA1) phosphorylation levels and increased glycogen synthase kinase 3 (GSK3) activity. Importantly, selective GSK3 inhibition using 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione increased levels of synapsin-1, BDNF and phosphorylated-GluA1 proteins, restored hippocampal basal synaptic transmission and LTP, and improved contextual fear memory in male Tg26 mice., Conclusion and Implications: Sex-dependent impairments in contextual fear memory and synaptic plasticity in Tg26 mice are associated with increased GSK3 activity. This implicates GSK3 inhibition as a potential therapeutic strategy to improve cognition in HIV-1 patients., (© 2020 The British Pharmacological Society.)

Clinical trials examining neuroprotective strategies after brain injury, including those targeting cell death mechanisms, have been underwhelming. This may be in part due to an incomplete understanding of the signalling mechanisms that induce cell death after traumatic brain injury. The recent identification of a new family of death receptors that initiate pro-cell death signals in the absence of their ligand, called dependence receptors, provides new insight into the factors that contribute to brain injury. Here, we show that blocking the dependence receptor signalling of EphB3 improves oligodendrocyte cell survival in a murine controlled cortical impact injury model, which leads to improved myelin sparing, axonal conductance and behavioural recovery. EphB3 also functions as a cysteine-aspartic protease substrate, where the recruitment of injury-dependent adaptor protein Dral/FHL-2 together with capsase-8 or -9 leads to EphB3 cleavage to initiate cell death signals in murine and human traumatic brain-injured patients, supporting a conserved mechanism of cell death. These pro-apoptotic responses can be blocked via exogenous ephrinB3 ligand administration leading to improved oligodendrocyte survival. In short, our findings identify a novel mechanism of oligodendrocyte cell death in the traumatically injured brain that may reflect an important neuroprotective strategy in patients., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Cognitive impairments are a common consequence of traumatic brain injury (TBI). The hippocampus is a subcortical structure that plays a key role in the formation of declarative memories and is highly vulnerable to TBI. The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in the hippocampus and reduced expression and function of this receptor are linked with cognitive impairments in Alzheimer's disease and schizophrenia. Positive allosteric modulation of α7 nAChRs with AVL-3288 enhances receptor currents and improves cognitive functioning in naïve animals and healthy human subjects. Therefore, we hypothesized that targeting the α7 nAChR with the positive allosteric modulator AVL-3288 would enhance cognitive functioning in the chronic recovery period of TBI. To test this hypothesis, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury or sham surgery. At 3 months after recovery, animals were treated with vehicle or AVL-3288 at 30 min prior to cue and contextual fear conditioning and the water maze task. Treatment of TBI animals with AVL-3288 rescued learning and memory deficits in water maze retention and working memory. AVL-3288 treatment also improved cue and contextual fear memory when tested at 24 hr and 1 month after training, when TBI animals were treated acutely just during fear conditioning at 3 months post-TBI. Hippocampal atrophy but not cortical atrophy was reduced with AVL-3288 treatment in the chronic recovery phase of TBI. AVL-3288 application to acute hippocampal slices from animals at 3 months after TBI rescued basal synaptic transmission deficits and long-term potentiation (LTP) in area CA1. Our results demonstrate that AVL-3288 improves hippocampal synaptic plasticity, and learning and memory performance after TBI in the chronic recovery period. Enhancing cholinergic transmission through positive allosteric modulation of the α7 nAChR may be a novel therapeutic to improve cognition after TBI., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: K.W.G., T.B.J. and D.J.H. are inventors on patents covering the compound AVL-3288 and uses thereof. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Traumatic brain injury (TBI) significantly decreases cyclic AMP (cAMP) signaling which produces long-term synaptic plasticity deficits and chronic learning and memory impairments. Phosphodiesterase 4 (PDE4) is a major family of cAMP hydrolyzing enzymes in the brain and of the four PDE4 subtypes, PDE4D in particular has been found to be involved in memory formation. Although most PDE4 inhibitors target all PDE4 subtypes, PDE4D can be targeted with a selective, negative allosteric modulator, D159687. In this study, we hypothesized that treating animals with D159687 could reverse the cognitive deficits caused by TBI. To test this hypothesis, adult male Sprague Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury. After 3 months of recovery, animals were treated with D159687 (0.3 mg/kg, intraperitoneally) at 30 min prior to cue and contextual fear conditioning, acquisition in the water maze or during a spatial working memory task. Treatment with D159687 had no significant effect on these behavioral tasks in non-injured, sham animals, but did reverse the learning and memory deficits in chronic TBI animals. Assessment of hippocampal slices at 3 months post-TBI revealed that D159687 reversed both the depression in basal synaptic transmission in area CA1 as well as the late-phase of long-term potentiation. These results demonstrate that a negative allosteric modulator of PDE4D may be a potential therapeutic to improve chronic cognitive dysfunction following TBI., (Copyright © 2018 Elsevier Inc. All rights reserved.)

With nearly 42 million mild traumatic brain injuries (mTBIs) occurring worldwide every year, understanding the factors that may adversely influence recovery after mTBI is important for developing guidelines in mTBI management. Extensive clinical evidence exists documenting the detrimental effects of elevated temperature levels on recovery after moderate to severe TBI. However, whether elevated temperature alters recovery after mTBI or concussion is an active area of investigation. Individuals engaged in exercise and competitive sports regularly experience body and brain temperature increases to hyperthermic levels and these temperature increases are prolonged in hot and humid ambient environments. Thus, there is a strong potential for hyperthermia to alter recovery after mTBI in a subset of individuals at risk for mTBI. Preclinical mTBI studies have found that elevating brain temperature to 39°C before mTBI significantly increases neuronal death within the cortex and hippocampus and also worsens cognitive deficits. This review summarizes the pathology and behavioral problems of mTBI that are exacerbated by hyperthermia and discusses whether hyperthermia is a variable that should be considered after concussion and mTBI. Finally, underlying pathophysiological mechanisms responsible for hyperthermia-induced altered responses to mTBI and potential gender considerations are discussed., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Traumatic brain injury (TBI) initiates a deleterious inflammatory response that exacerbates pathology and worsens outcome. This inflammatory response is partially mediated by a reduction in cAMP and a concomitant upregulation of cAMP-hydrolyzing phosphodiesterases (PDEs) acutely after TBI. The PDE4B subfamily, specifically PDE4B2, has been found to regulate cAMP in inflammatory cells, such as neutrophils, macrophages and microglia. To determine if PDE4B regulates inflammation and subsequent pathology after TBI, adult male Sprague Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury (2 ± 0.2 atm) and were then treated with a PDE4B - selective inhibitor, A33, or vehicle for up to 3 days post-surgery. Treatment with A33 reduced markers of microglial activation and neutrophil infiltration at 3 and 24 hrs after TBI, respectively. A33 treatment also reduced cortical contusion volume at 3 days post-injury. To determine whether this treatment paradigm attenuated TBI-induced behavioral deficits, animals were evaluated over a period of 6 weeks after surgery for forelimb placement asymmetry, contextual fear conditioning, water maze performance and spatial working memory. A33 treatment significantly improved contextual fear conditioning and water maze retention at 24 hrs post-training. However, this treatment did not rescue sensorimotor or working memory deficits. At 2 months after surgery, atrophy and neuronal loss were measured. A33 treatment significantly reduced neuronal loss in the pericontusional cortex and hippocampal CA3 region. This treatment paradigm also reduced cortical, but not hippocampal, atrophy. Overall, these results suggest that acute PDE4B inhibition may be a viable treatment to reduce inflammation, pathology and memory deficits after TBI.

Unlabelled: Learning and memory impairments are common in traumatic brain injury (TBI) survivors. However, there are no effective treatments to improve TBI-induced learning and memory impairments. TBI results in decreased cAMP signaling and reduced cAMP-response-element binding protein (CREB) activation, a critical pathway involved in learning and memory. TBI also acutely upregulates phosphodiesterase 4B2 (PDE4B2), which terminates cAMP signaling by hydrolyzing cAMP. We hypothesized that a subtype-selective PDE4B inhibitor could reverse the learning deficits induced by TBI. To test this hypothesis, adult male Sprague-Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury. At 3 months postsurgery, animals were administered a selective PDE4B inhibitor or vehicle before cue and contextual fear conditioning, water maze training and a spatial working memory task. Treatment with the PDE4B inhibitor significantly reversed the TBI-induced deficits in cue and contextual fear conditioning and water maze retention. To further understand the underlying mechanisms of these memory impairments, we examined hippocampal long-term potentiation (LTP). TBI resulted in a significant reduction in basal synaptic transmission and impaired expression of LTP. Treatment with the PDE4B inhibitor significantly reduced the deficits in basal synaptic transmission and rescued LTP expression. The PDE4B inhibitor reduced tumor necrosis factor-α levels and increased phosphorylated CREB levels after TBI, suggesting that this drug inhibited molecular pathways in the brain known to be regulated by PDE4B. These results suggest that a subtype-selective PDE4B inhibitor is a potential therapeutic to reverse chronic learning and memory dysfunction and deficits in hippocampal synaptic plasticity following TBI., Significance Statement: Currently, there are an estimated 3.2-5.3 million individuals living with disabilities from traumatic brain injury (TBI) in the United States, and 8 of 10 of these individuals report cognitive disabilities (Thurman et al., 1999; Lew et al., 2006; Zaloshnja et al., 2008). One of the molecular mechanisms associated with chronic cognitive disabilities is impaired cAMP signaling in the hippocampus. In this study, we report that a selective phosphodiesterase 4B (PDE4B) inhibitor reduces chronic cognitive deficits after TBI and rescues deficits in hippocampal long-term potentiation. These results suggest that PDE4B inhibition has the potential to improve learning and memory ability and overall functioning for people living with TBI., (Copyright © 2016 the authors 0270-6474/16/367095-14$15.00/0.)

Traumatic brain injury (TBI) results in significant impairments in hippocampal synaptic plasticity. A molecule critically involved in hippocampal synaptic plasticity, 3',5'-cyclic adenosine monophosphate, is downregulated in the hippocampus after TBI, but the mechanism that underlies this decrease is unknown. To address this question, we determined whether phosphodiesterase (PDE) expression in the hippocampus is altered by TBI. Young adult male Sprague Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury. Animals were analyzed by western blotting for changes in PDE expression levels in the hippocampus. We found that PDE1A levels were significantly increased at 30 min, 1 h and 6 h after TBI. PDE4B2 and 4D2 were also significantly increased at 1, 6, and 24 h after TBI. Additionally, phosphorylation of PDE4A was significantly increased at 6 and 24 h after TBI. No significant changes were observed in levels of PDE1B, 1C, 3A, 8A, or 8B between 30 min to 7 days after TBI. To determine the spatial profile of these increases, we used immunohistochemistry and flow cytometry at 24 h after TBI. PDE1A and phospho-PDE4A localized to neuronal cell bodies. PDE4B2 was expressed in neuronal dendrites, microglia and infiltrating CD11b(+) immune cells. PDE4D was predominantly found in microglia and infiltrating CD11b(+) immune cells. To determine if inhibition of PDE4 would improve hippocampal synaptic plasticity deficits after TBI, we treated hippocampal slices with rolipram, a pan-PDE4 inhibitor. Rolipram partially rescued the depression in basal synaptic transmission and converted a decaying form of long-term potentiation (LTP) into long-lasting LTP. Overall, these results identify several possible PDE targets for reducing hippocampal synaptic plasticity deficits and improving cognitive function acutely after TBI.

Mild elevations in core temperature can occur in individuals involved in strenuous activities that are risky for potentially sustaining a mild traumatic brain injury (mTBI) or concussion. Recently, we have discovered that mild elevations in brain temperature can significantly aggravate the histopathological consequences of mTBI. However, whether this exacerbation of brain pathology translates into behavioral deficits is unknown. Therefore, we investigated the behavioral consequences of elevating brain temperature to mildly hyperthermic levels prior to mTBI. Adult male Sprague Dawley rats underwent mild fluid-percussion brain injury or sham surgery while normothermic (37 °C) or hyperthermic (39 °C) and were allowed to recover for 7 days. Animals were then assessed for cognition using the water maze and cue and contextual fear conditioning. We found that mTBI alone at normothermia had no effect on long-term cognitive measures whereas mTBI animals that were hyperthermic for 15 min prior to and for 4h after brain injury were significantly impaired on long-term retention for both the water maze and fear conditioning. In contrast, hyperthermic mTBI animals cooled within 15 min to normothermia demonstrated no significant long-term cognitive deficits. Mild TBI irrespective of temperature manipulations resulted in significant short-term working memory deficits. Cortical atrophy and contusions were detected in all mTBI treatment groups and contusion volume was significantly less in hyperthermic mTBI animals that were cooled as compared to hyperthermic mTBI animals that remained hyperthermic. These results indicate that brain temperature is an important variable for mTBI outcome and that mildly elevated temperatures at the time of injury result in persistent cognitive deficits. Importantly, cooling to normothermia after mTBI prevents the development of long-term cognitive deficits caused by hyperthermia. Reducing temperature to normothermic levels soon after mTBI represents a rational approach to potentially mitigate the long-term consequences of mTBI., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Developing therapeutics for traumatic brain injury remains a challenge for all stages of recovery. The pathological features of traumatic brain injury are diverse, and it remains an obstacle to be able to target the wide range of pathologies that vary between traumatic brain injured patients and that evolve during recovery. One promising therapeutic avenue is to target the second messengers cAMP and cGMP with phosphodiesterase inhibitors due to their broad effects within the nervous system. Phosphodiesterase inhibitors have the capability to target different injury mechanisms throughout the time course of recovery after brain injury. Inflammation and neuronal death are early targets of phosphodiesterase inhibitors, and synaptic dysfunction and circuitry remodeling are late potential targets of phosphodiesterase inhibitors. This review will discuss how signaling through cyclic nucleotides contributes to the pathology of traumatic brain injury in the acute and chronic stages of recovery. We will review our current knowledge of the successes and challenges of using phosphodiesterase inhibitors for the treatment of traumatic brain injury and conclude with important considerations in developing phosphodiesterase inhibitors as therapeutics for brain trauma.

Traumatic brain injury (TBI) modulates several cell signaling pathways in the hippocampus critical for memory formation. Previous studies have found that the cAMP-protein kinase A signaling pathway is downregulated after TBI and that treatment with a phosphodiesterase (PDE) 4 inhibitor rolipram rescues the decrease in cAMP. In the present study, we examined the effect of rolipram on TBI-induced cognitive impairments. At 2 weeks after moderate fluid-percussion brain injury or sham surgery, adult male Sprague Dawley rats received vehicle or rolipram (0.03 mg/kg) 30 min before water maze acquisition or cue and contextual fear conditioning. TBI animals treated with rolipram showed a significant improvement in water maze acquisition and retention of both cue and contextual fear conditioning compared with vehicle-treated TBI animals. Cue and contextual fear conditioning significantly increased phosphorylated CREB levels in the hippocampus of sham animals, but not in TBI animals. This deficit in CREB activation during learning was rescued in TBI animals treated with rolipram. Hippocampal long-term potentiation was reduced in TBI animals, and this was also rescued with rolipram treatment. These results indicate that the PDE4 inhibitor rolipram rescues cognitive impairments after TBI, and this may be mediated through increased CREB activation during learning.

The elderly have comparatively worse cognitive impairments from traumatic brain injury (TBI) relative to younger adults, but the molecular mechanisms that underlie this exacerbation of cognitive deficits are unknown. Experimental models of TBI have demonstrated that the cyclic AMP-protein kinase A (cAMP-PKA) signaling pathway is downregulated after brain trauma. Since the cAMP-PKA signaling pathway is a key mediator of long-term memory formation, we investigated whether the TBI-induced decrease in cAMP levels is exacerbated in aged animals. Aged (19 months) and young adult (3 months) male Fischer 344 rats received sham surgery or mild (1.4-1.6 atmospheres, atm) or moderate (1.7-2.1 atm) parasagittal fluid-percussion brain injury. At various time points after surgery, the ipsilateral parietal cortex, hippocampus, and thalamus were assayed for cAMP levels. Mild TBI lowered cAMP levels in the hippocampus of aged, but not young adult animals. Moderate TBI lowered cAMP levels in the hippocampus and parietal cortex of both age groups. In the thalamus, cAMP levels were significantly lowered after moderate, but not mild TBI. To determine if the TBI-induced decreases in cAMP had physiological consequences in aged animals, hippocampal long-term potentiation (LTP) in the Schaffer collateral pathway of the CA1 region was assessed. LTP was significantly decreased in both young adult and aged animals after mild and moderate TBI as compared to sham surgery animals. Rolipram rescued the LTP deficits after mild TBI for young adult animals and caused a partial recovery for aged animals. However, rolipram did not rescue LTP deficits after moderate TBI in either young adult or aged animals. These results indicate that the exacerbation of cognitive impairments in aged animals with TBI may be due to decreased cAMP levels and deficits in hippocampal LTP., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Traumatic brain injury (TBI) pathology includes contusions, cavitation, cell death, all of which can be exacerbated by inflammation. We hypothesized that an anti-inflammatory drug, rolipram, may reduce pathology after TBI, since in several CNS injury models rolipram reduces inflammation and improves cell survival and functional recovery. Adult male C57BL/6 mice received a craniotomy over the right parietotemporal cortex. Vertically directed controlled cortical impact (CCI) injury was delivered. Naïve controls were used for comparison. At 30 min post-surgery, animals were treated with vehicle or rolipram (1 mg/kg), and then once per day for 3 days. On day 3, the brains were systematically sectioned and stained to visualize the resulting pathology using hematoxylin and eosin (H&E) staining and NeuN immunocytochemistry. Total parietotemporal cortical contusion and cavity volume were significantly increased in rolipram-treated as compared to vehicle-treated CCI animals. Contusion areas at specific bregma levels indicated a significant effect of drug across bregma levels. Neuronal cell loss in the dentate hilus and area CA3 of the hippocampus were similar between vehicle and rolipram-treated animals. Although rolipram is well known to reduce pathology and inflammation in several other CNS injury models, the pathology resulting from CCI was worsened with rolipram at this particular dose and administration schedule. These studies suggest that consideration of the unique characteristics of TBI pathology is important in the extrapolation of promising therapeutic interventions from other CNS injury models., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Traumatic brain injury (TBI) results in significant inflammation which contributes to the evolving pathology. Previously, we have demonstrated that cyclic AMP (cAMP), a molecule involved in inflammation, is down-regulated after TBI. To determine the mechanism by which cAMP is down-regulated after TBI, we determined whether TBI induces changes in phosphodiesterase (PDE) expression. Adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury (FPI) or sham injury, and the ipsilateral, parietal cortex was analyzed by western blotting. In the ipsilateral parietal cortex, expression of PDE1A, PDE4B2, and PDE4D2, significantly increased from 30 min to 24 h post-injury. PDE10A significantly increased at 6 and 24 h after TBI. Phosphorylation of PDE4A significantly increased from 6 h to 7 days post-injury. In contrast, PDE1B, PD4A5, and PDE4A8 significantly decreased after TBI. No changes were observed with PDE1C, PDE3A, PDE4B1/3, PDE4B4, PDE4D3, PDE4D4, PDE8A, or PDE8B. Co-localization studies showed that PDE1A, PDE4B2, and phospho-PDE4A were neuronally expressed, whereas PDE4D2 was expressed in neither neurons nor glia. These findings suggest that therapies to reduce inflammation after TBI could be facilitated with targeted therapies, in particular for PDE1A, PDE4B2, PDE4D2, or PDE10A., (© 2012 International Society for Neurochemistry.)

Cyclic AMP suppresses immune cell activation and inflammation. The positive feedback loop of proinflammatory cytokine production and immune activation implies that cytokines may not only be regulated by cyclic AMP but also conversely regulate cyclic AMP. This study examined the effects of tumor necrosis factor (TNF)-α and interleukin (IL)-1β on cyclic AMP-phosphodiesterase (PDE) signaling in microglia in vitro and after spinal cord injury (SCI) or traumatic brain injury (TBI). TNF-α or IL-1β stimulation produced a profound reduction (>90%) of cyclic AMP within EOC2 microglia from 30 min that then recovered after IL-1β but remained suppressed with TNF-α through 24 h. Cyclic AMP was also reduced in TNF-α-stimulated primary microglia, albeit to a lesser extent. Accompanying TNF-α-induced cyclic AMP reductions, but not IL-1β, was increased cyclic AMP-PDE activity. The role of PDE4 activity in cyclic AMP reductions was confirmed by using Rolipram. Examination of pde4 mRNA revealed an immediate, persistent increase in pde4b with TNF-α; IL-1β increased all pde4 mRNAs. Immunoblotting for PDE4 showed that both cytokines increased PDE4A1, but only TNF-α increased PDE4B2. Immunocytochemistry revealed PDE4B nuclear translocation with TNF-α but not IL-1β. Acutely after SCI/TBI, where cyclic AMP levels are reduced, PDE4B was localized to activated OX-42(+) microglia; PDE4B was absent in OX-42(+) cells in uninjured spinal cord/cortex or inactive microglia. Immunoblotting showed PDE4B2 up-regulation from 24 h to 1 wk post-SCI, the peak of microglia activation. These studies show that TNF-α and IL-1β differentially affect cyclic AMP-PDE signaling in microglia. Targeting PDE4B2 may be a putative therapeutic direction for reducing microglia activation in CNS injury and neurodegenerative diseases., (Copyright © 2012 Wiley Periodicals, Inc.)

The pathology caused by traumatic brain injury (TBI) is exacerbated by the inflammatory response of the injured brain. Two proinflammatory cytokines that contribute to inflammation after TBI are tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). From previous studies using the parasagittal fluid-percussion brain injury model, we reported that the anti-inflammatory drug rolipram, a phosphodiesterase 4 inhibitor, reduced TNF-α and IL-1β levels and improved histopathological outcome when administered 30 min prior to injury. We now report that treatment with (±)-rolipram given 30 min after injury significantly reduced TNF-α levels in the cortex and hippocampus. However, postinjury administration of (±)-rolipram significantly increased cortical contusion volume and increased atrophy of the cortex compared with vehicle-treated animals at 10 days postinjury. Thus, despite the reduction in proinflammatory cytokine levels, histopathological outcome was worsened with post-TBI (±)-rolipram treatment. Further histological analysis of (±)-rolipram-treated TBI animals revealed significant hemorrhage in the contused brain. Given the well-known role of (±)-rolipram of increasing vasodilation, it is likely that (±)-rolipram worsened outcome after fluid-percussion brain injury by causing increased bleeding., (Copyright © 2012 Wiley Periodicals, Inc.)

Astrocytes respond to trauma by stimulating inflammatory signaling. In studies of cerebral ischemia and spinal cord injury, astrocytic signaling is mediated by the cytokine receptor glycoprotein 130 (gp130) and Janus kinase (Jak) which phosphorylates the transcription factor signal transducer and activator of transcription-3 (STAT3). To determine if STAT3 is activated after traumatic brain injury (TBI), adult male Sprague-Dawley rats received moderate parasagittal fluid-percussion brain injury or sham surgery, and then the ipsilateral cortex and hippocampus were analyzed at various post-traumatic time periods for up to 7 days. Western blot analyses indicated that STAT3 phosphorylation significantly increased at 30 min and lasted for 24 h post-TBI. A significant increase in gp130 and Jak2 phosphorylation was also observed. Confocal microscopy revealed that STAT3 was localized primarily within astrocytic nuclei. At 6 and 24 h post-TBI, there was also an increased expression of STAT3 pathway-related genes: suppressor of cytokine signaling 3, nitric oxide synthase 2, colony stimulating factor 2 receptor β, oncostatin M, matrix metalloproteinase 3, cyclin-dependent kinase inhibitor 1A, CCAAT/enhancer-binding protein β, interleukin-2 receptor γ, interleukin-4 receptor α, and α-2-macroglobulin. These results clarify some of the signaling pathways operative in astrocytes after TBI and demonstrate that the gp130-Jak2-STAT3 signaling pathway is activated after TBI in astrocytes., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Previous studies have demonstrated that moderate hypothermia reduces histopathological damage and improves behavioral outcome after experimental traumatic brain injury (TBI). Further investigations have clarified the mechanisms underlying the beneficial effects of hypothermia by showing that cooling reduces multiple cell injury cascades. The purpose of this study was to determine whether hypothermia could also enhance endogenous reparative processes following TBI such as neurogenesis and the replacement of lost neurons. Male Sprague-Dawley rats underwent moderate fluid-percussion brain injury and then were randomized into normothermia (37°C) or hypothermia (33°C) treatment. Animals received injections of 5-bromo-2'-deoxyuridine (BrdU) to detect mitotic cells after brain injury. After 3 or 7 days, animals were perfusion-fixed and processed for immunocytochemistry and confocal analysis. Sections were stained for markers selective for cell proliferation (BrdU), neuroblasts and immature neurons (doublecortin), and mature neurons (NeuN) and then analyzed using non-biased stereology to quantify neurogenesis in the dentate gyrus (DG). At 7 days after TBI, both normothermic and hypothermic TBI animals demonstrated a significant increase in the number of BrdU-immunoreactive cells in the DG as compared to sham-operated controls. At 7 days post-injury, hypothermia animals had a greater number of BrdU (ipsilateral cortex) and doublecortin (ipsilateral and contralateral cortex) immunoreactive cells in the DG as compared to normothermia animals. Because adult neurogenesis following injury may be associated with enhanced functional recovery, these data demonstrate that therapeutic hypothermia sustains the increase in neurogenesis induced by TBI and this may be one of the mechanisms by which hypothermia promotes reparative strategies in the injured nervous system., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The effects of slight variations in brain temperature on the pathophysiological consequences of acute brain injury have been extensively described in models of moderate and severe traumatic brain injury (TBI). In contrast, limited information is available regarding the potential consequences of temperature elevations on outcome following mild TBI (mTBI) or concussions. One potential confounding variable with mTBI is the presence of elevated body temperature that occurs in the civilian or military populations due to hot environments combined with exercise or other forms of physical exertion. We therefore determined the histopathological effects of pre- and post-traumatic hyperthermia (39°C) on mTBI. Adult male Sprague-Dawley rats were divided into 3 groups: pre/post-traumatic hyperthermia, post-traumatic hyperthermia alone for 2 h, and normothermia (37°C). The pre/post-hyperthermia group was treated with hyperthermia starting 15 min before mild parasagittal fluid-percussion brain injury (1.4-1.6 atm), with the temperature elevation extending for 2 h after trauma. At 72 h after mTBI, the rats were perfusion-fixed for quantitative histopathological evaluation. Contusion areas and volumes were significantly larger in the pre/post-hyperthermia treatment group compared to the post-hyperthermia and normothermic groups. In addition, pre/post-traumatic hyperthermia caused the most severe loss of NeuN-positive cells in the dentate hilus compared to normothermia. These neuropathological results demonstrate that relatively mild elevations in temperature associated with peri-traumatic events may affect the long-term functional consequences of mTBI. Because individuals exhibiting mildly elevated core temperatures may be predisposed to aggravated brain damage after mTBI or concussion, precautions should be introduced to target this important physiological variable.

There are more than 3.17 million people coping with long-term disabilities due to traumatic brain injury (TBI) in the United States. The majority of TBI research is focused on developing acute neuroprotective treatments to prevent or minimize these long-term disabilities. Therefore, chronic TBI survivors represent a large, underserved population that could significantly benefit from a therapy that capitalizes on the endogenous recovery mechanisms occurring during the weeks to months following brain trauma. Previous studies have found that the hippocampus is highly vulnerable to brain injury, in both experimental models of TBI and during human TBI. Although often not directly mechanically injured by the head injury, in the weeks to months following TBI, the hippocampus undergoes atrophy and exhibits deficits in long-term potentiation (LTP), a persistent increase in synaptic strength that is considered to be a model of learning and memory. Decoding the chronic hippocampal LTP and cell signaling deficits after brain trauma will provide new insights into the molecular mechanisms of hippocampal-dependent learning impairments caused by TBI and facilitate the development of effective therapeutic strategies to improve hippocampal-dependent learning for chronic survivors of TBI.

Edema, the accumulation of excess fluid, is a major pathological change in the brain that contributes significantly to pathology and mortality after moderate to severe brain injury. Edema is regulated by aquaporin (AQP) channels which transport water across cellular membranes. Six AQPs are found in the brain (1, 3, 4, 5, 8, and 9), and previous studies have found that AQP4 is regulated after traumatic brain injury (TBI). To further understand how AQPs contribute to brain edema, we investigated whether expression of AQP1, 3, and 9 are also regulated after TBI. Adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury (FPI) or sham surgery. After induction of FPI, the injured, ipsilateral parietal cortex and hippocampus were dissected and analyzed by Western blotting. We observed a small decrease in AQP3 and 4 levels at 7 days after FPI in the ipsilateral, parietal cortex. Both AQP1 and 9 significantly increased within 30 min post-injury and remained elevated for up to 6 h in the ipsilateral, parietal cortex. Aqp1 and 9 mRNA levels were also significantly increased at 30 min post-FPI. Administration of an AQP1 and 4 antagonist, AqB013, non-significantly increased brain water content in sham, non-injured animals, and did not prevent edema formation 24 h after trauma in either the parietal cortex or hippocampus. These results indicate that Aqp1 and 9 mRNA and protein levels increase after moderate parasagittal FPI and that an inhibitor of AQP1 and 4 does not decrease edema after moderate parasagittal FPI., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The purpose of this study was to investigate the effects of an induced period of post-traumatic epilepsy (PTE) on the histopathological damage caused by traumatic brain injury (TBI). Male Sprague Dawley rats were given a moderate parasagittal fluid-percussion brain injury (1.9-2.1 atm) or sham surgery. At 2 weeks after surgery, seizures were induced by administration of a GABA(A) receptor antagonist, pentylenetetrazole (PTZ, 30 mg/kg). Seizures were then assessed over a 1-h period using the Racine clinical rating scale. To evaluate whether TBI-induced pathology was exacerbated by the seizures, contusion volume and cortical and hippocampal CA3 neuronal cell loss were measured 3 days after seizures. Nearly all TBI rats showed clinical signs of PTE following the decrease in inhibitory activity. In contrast, clinically evident seizures were not observed in TBI rats given saline or sham-operated rats given PTZ. Contusions in TBI-PTZ-treated rats were significantly increased compared to the TBI-saline-treated group (p < 0.001). In addition, the TBI-PTZ rats showed less NeuN-immunoreactive cells within the ipsilateral parietal cerebral cortex (p < 0.05) and there was a trend for decreased hippocampal CA3 neurons in TBI-PTZ rats compared with TBI-saline or sham-operated rats. These results demonstrate that an induced period of post-traumatic seizures significantly exacerbates the structural damage caused by TBI. These findings emphasize the need to control seizures after TBI to limit even further damage to the injured brain.

Traumatic brain injury (TBI) is a major risk factor for the subsequent development of epilepsy. Currently, chronic seizures after brain injury are often poorly controlled by available antiepileptic drugs. Hypothermia treatment, a modest reduction in brain temperature, reduces inflammation, activates pro-survival signaling pathways, and improves cognitive outcome after TBI. Given the well-known effect of therapeutic hypothermia to ameliorate pathological changes in the brain after TBI, we hypothesized that hypothermia therapy may attenuate the development of post-traumatic epilepsy and some of the pathomechanisms that underlie seizure formation. To test this hypothesis, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury, and were then maintained at normothermic or moderate hypothermic temperatures for 4 h. At 12 weeks after recovery, seizure susceptibility was assessed by challenging the animals with pentylenetetrazole, a GABA(A) receptor antagonist. Pentylenetetrazole elicited a significant increase in seizure frequency in TBI normothermic animals as compared with sham surgery animals and this was significantly reduced in TBI hypothermic animals. Early hypothermia treatment did not rescue chronic dentate hilar neuronal loss nor did it improve loss of doublecortin-labeled cells in the dentate gyrus post-seizures. However, mossy fiber sprouting was significantly attenuated by hypothermia therapy. These findings demonstrate that reductions in seizure susceptibility after TBI are improved with post-traumatic hypothermia and provide a new therapeutic avenue for the treatment of post-traumatic epilepsy., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Traumatic brain injury (TBI) activates several protein kinase signaling pathways in the hippocampus that are critical for hippocampal-dependent memory formation. In particular, extracellular signal-regulated kinase (ERK), a protein kinase activated during and necessary for hippocampal-dependent learning, is transiently activated after TBI. However, TBI patients experience hippocampal-dependent cognitive deficits that occur for several months to years after the initial injury. Although basal activation levels of ERK return to sham levels within hours after TBI, we hypothesized that activation of ERK may be impaired after TBI. Adult male Sprague-Dawley rats received either sham surgery or moderate parasagittal fluid-percussion brain injury. At 2, 8, or 12 weeks after surgery, the ipsilateral hippocampi of sham surgery and TBI animals were sectioned into transverse slices. After 2h of recovery in oxygenated artificial cerebrospinal fluid, the hippocampal slices were stimulated with glutamate or KCl depolarization, then analyzed by western blotting for phosphorylated, activated ERK and one of its downstream effectors, the transcription factor cAMP response element-binding protein (CREB). We found that activation of ERK (p<0.05) and CREB (p<0.05) after 30s of glutamate stimulation or KCl depolarization was decreased in hippocampal slices from animals at 2, 8, or 12 weeks after TBI as compared to sham animals. Basal levels of phosphorylated or total ERK were not significantly altered at 2, 8, or 12 weeks after TBI, although basal levels of phosphorylated CREB were decreased 12 weeks post-trauma. These results suggest that TBI results in chronic signaling deficits through the ERK-CREB pathway in the hippocampus.

For the past 20 years, various laboratories throughout the world have shown that mild to moderate levels of hypothermia lead to neuroprotection and improved functional outcome in various models of brain and spinal cord injury (SCI). Although the potential neuroprotective effects of profound hypothermia during and following central nervous system (CNS) injury have long been recognized, more recent studies have described clinically feasible strategies for protecting the brain and spinal cord using hypothermia following a variety of CNS insults. In some cases, only a one or two degree decrease in brain or core temperature can be effective in protecting the CNS from injury. Alternatively, raising brain temperature only a couple of degrees above normothermia levels worsens outcome in a variety of injury models. Based on these data, resurgence has occurred in the potential use of therapeutic hypothermia in experimental and clinical settings. The study of therapeutic hypothermia is now an international area of investigation with scientists and clinicians from every part of the world contributing to this important, promising therapeutic intervention. This paper reviews the experimental data obtained in animal models of brain and SCI demonstrating the benefits of mild to moderate hypothermia. These studies have provided critical data for the translation of this therapy to the clinical arena. The mechanisms underlying the beneficial effects of mild hypothermia are also summarized.

Traumatic brain injury (TBI) results in both focal and diffuse brain pathologies that are exacerbated by the inflammatory response and progress from hours to days after the initial injury. Using a clinically relevant model of TBI, the parasagittal fluid-percussion brain injury (FPI) model, we found injury-induced impairments in the cyclic AMP (cAMP) signaling pathway. Levels of cAMP were depressed in the ipsilateral parietal cortex and hippocampus, as well as activation of its downstream target, protein kinase A, from 15 min to 48 h after moderate FPI. To determine if preventing hydrolysis of cAMP by administration of a phosphodiesterase (PDE) IV inhibitor would improve outcome after TBI, we treated animals intraperitoneally with rolipram (0.3 or 3.0 mg/kg) 30 min prior to TBI, and then once per day for 3 days. Rolipram treatment restored cAMP to sham levels and significantly reduced cortical contusion volume and improved neuronal cell survival in the parietal cortex and CA3 region of the hippocampus. Traumatic axonal injury, characterized by beta-amyloid precursor protein deposits in the external capsule, was also significantly reduced in rolipram-treated animals. Furthermore, levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), were significantly decreased with rolipram treatment. These results demonstrate that the cAMP-PKA signaling cascade is downregulated after TBI, and that treatment with a PDE IV inhibitor improves histopathological outcome and decreases inflammation after TBI.

Traumatic brain injury (TBI) results in significant hippocampal pathology and hippocampal-dependent memory loss, both of which are alleviated by hypothermia treatment. To elucidate the molecular mechanisms regulated by hypothermia after TBI, rats underwent moderate parasagittal fluid-percussion brain injury. Brain temperature was maintained at normothermic or hypothermic temperatures for 30 min prior and up to 4 h after TBI. The ipsilateral hippocampus was assayed with Western blotting. We found that hypothermia potentiated extracellular signal-regulated kinase 1/2 (ERK1/2) activation and its downstream effectors, p90 ribosomal S6 kinase (p90RSK) and the transcription factor cAMP response element-binding protein. Phosphorylation of another p90RSK substrate, Bad, also increased with hypothermia after TBI. ERK1/2 regulates mRNA translation through phosphorylation of mitogen-activated protein kinase-interacting kinase 1 (Mnk1) and the translation factor eukaryotic initiation factor 4E (eIF4E). Hypothermia also potentiated the phosphorylation of both Mnk1 and eIF4E. Augmentation of ERK1/2 activation and its downstream signalling components may be one molecular mechanism that hypothermia treatment elicits to improve functional outcome after TBI.

In response to traumatic brain injury (TBI), neurons initiate neuroplastic processes through the activation of intracellular signaling pathways. However, the molecular mechanisms underlying neuroplasticity after TBI are poorly understood. To study this, we utilized the fluid-percussion brain injury (FPI) model to investigate alterations in the mammalian target of rapamycin (mTOR) signaling pathways in response to TBI. Mammalian target of rapamycin stimulates mRNA translation through phosphorylation of eukaryotic initiation factor 4E binding protein-1 (4E-BP1), p70 ribosomal S6 kinase (p70S6K), and ribosomal protein S6 (rpS6). These pathways coordinate cell growth and neuroplasticity via dendritic protein synthesis. Rats received sham surgery or moderate parasagittal FPI on the right side of the parietal cortex, followed by 15 mins, 30 mins, 4 h, 24 h, or 72 h of recovery. Using Western blot analysis, we found that mTOR, p70S6K, rpS6, and 4E-BP1 phosphorylation levels were significantly increased in the ipsilateral parietal cortex and hippocampus from 30 mins to 24 h after TBI, whereas total protein levels were unchanged. Using confocal microscopy to localize these changes, we found that rpS6 phosphorylation was increased in the parietal cortex and all subregions of the hippocampus. In accordance with these results, eIF4E, a key, rate-limiting mRNA translation factor, was also phosphorylated by mitogen-activated protein kinase-interacting kinase 1 (Mnk1) 15 mins after TBI. Together, these results suggest that changes in mRNA translation may be one mechanism that neurons use to respond to trauma and may contribute to the neuroplastic changes observed after TBI.

A prominent cognitive impairment after traumatic brain injury (TBI) is hippocampal-dependent memory loss. Although the histopathologic changes in the brain are well documented after TBI, the underlying biochemical mechanisms that contribute to memory loss have yet to be thoroughly delineated. Thus, we determined if calcium/calmodulin-dependent protein kinases (CaMKs), known to be necessary for the formation of hippocampal-dependent memories, are regulated after TBI. Sprague-Dawley rats underwent moderate parasagittal fluid-percussion brain injury on the right side of the parietal cortex. The ipsilateral hippocampus and parietal cortex were Western blotted for phosphorylated, activated alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaMKII), CaMKIV, and CaMKI. alpha-Calcium/calmodulin-dependent protein kinase II was activated in membrane subcellular fractions from the hippocampus and parietal cortex 30 mins after TBI. CaMKI and CaMKIV were activated in a more delayed manner, increasing in phosphorylation 1 h after TBI. The increase in activated alpha-CaMKII in membrane fractions was accompanied by a decrease in cytosolic total alpha-CaMKII, suggesting redistribution to the membrane. Using confocal microscopy, we observed that alpha-CaMKII was activated within hippocampal neurons of the dentate gyrus, CA3, and CA1 regions. Two downstream substrates of alpha-CaMKII, the AMPA-type glutamate receptor GluR1, and cytoplasmic polyadenylation element-binding protein, concomitantly increased in phosphorylation in the hippocampus and cortex 1 h after TBI. These results demonstrate that several of the biochemical cascades that subserve memory formation are activated unselectively in neurons after TBI. As memory formation requires activation of CaMKII signaling pathways at specific neuronal synapses, unselective activation of CaMKII signaling in all synapses may disrupt the machinery for memory formation, resulting in memory loss after TBI.

A critical transition in neuron development is formation of the axon, which establishes the polarized structure of the neuron that underlies its entire input and output capabilities. The morphological events that occur during axonogenesis have long been known, yet the molecular determinants underlying axonogenesis remain poorly understood. We demonstrate here that axonogenesis requires activated c-Jun N-terminal kinase (JNK). JNK is expressed throughout the neuron, but its phosphorylated, activated form is highly enriched in the axon. In young axons, activated JNK forms a proximodistal gradient of increasing intensity, beginning at about the point where the axon exceeds the lengths of the other neurites (minor processes). Treatment with SP600125, a specific inhibitor of JNK, reversibly inhibits axonogenesis but does not prevent the formation of minor processes or their differentiation into dendrites (based on their immunostaining with marker proteins). Expression of a dominant-negative construct against JNK similarly prevents axonogenesis. Investigation of JNK targets revealed that activating transcription factor-2 is phosphorylated under normal conditions in neurons, and its phosphorylation is significantly attenuated after JNK inhibition. These results demonstrate that activated JNK is required for axonogenesis but not formation of minor processes or development of dendrites.

Induction of hippocampal long-term potentiation (LTP) requires activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), whereas maintenance of LTP additionally requires protein synthesis. We recently reported that CaMKII stimulates protein synthesis in depolarized hippocampal neurons through phosphorylation of the mRNA translation factor cytoplasmic polyadenylation element-binding protein (CPEB), and this phosphorylation is rapidly reversed by protein phosphatase 1 (PP1). Protein synthesis-dependent late-phase LTP (L-LTP) in the hippocampus requires calcium influx through the NMDA-type glutamate receptor (NMDA-R) to activate CaMKII as well as concomitant inhibition of PP1 mediated by protein kinase A. Therefore, we investigated the regulation of CPEB phosphorylation during L-LTP. Pharmacological stimulation of the NMDA-R in hippocampal slices to produce chemical long-term depression induced a brief dephosphorylation of CPEB. Modest LTP induction (once at 100 Hz), which induces a protein synthesis-independent early-phase LTP (E-LTP), resulted in a transient phosphorylation of CPEB. However, stronger stimulation (four times at 100 Hz), known to induce protein synthesis-dependent L-LTP, elicited a prolonged phosphorylation of CPEB. Furthermore, CPEB phosphorylation correlated with phosphorylation of PP1 inhibitor dopamine- and cAMP-regulated phosphoprotein, a known substrate for protein kinase A. These results evoke the hypothesis that bidirectional regulation of CPEB phosphorylation by CaMKII and protein phosphatases may serve as a mechanism to convert E-LTP into protein synthesis-dependent L-LTP by stimulating protein synthesis and thereby stabilizing synaptic enhancement.

Phosphorylation of cytoplasmic polyadenylation element binding protein (CPEB) regulates protein synthesis in hippocampal dendrites. CPEB binds the 3' untranslated region (UTR) of cytoplasmic mRNAs and, when phosphorylated, initiates mRNA polyadenylation and translation. We report that, of the protein kinases activated in the hippocampus during synaptic plasticity, calcium/calmodulin-dependent protein kinase II (CaMKII) robustly phosphorylated the regulatory site (threonine 171) in CPEB in vitro. In postsynaptic density fractions or hippocampal neurons, CPEB phosphorylation increased when CaMKII was activated. These increases in CPEB phosphorylation were attenuated by a specific peptide inhibitor of CaMKII and by the general CaM-kinase inhibitor KN-93. Inhibitors of protein phosphatase 1 increased basal CPEB phosphorylation in neurons; this was also attenuated by a CaM-kinase inhibitor. To determine whether CaM-kinase activity regulates CPEB-dependent mRNA translation, hippocampal neurons were transfected with luciferase fused to a 3' UTR containing CPE-binding elements. Depolarization of neurons stimulated synthesis of luciferase; this was abrogated by inhibitors of protein synthesis, mRNA polyadenylation, and CaMKII. These results demonstrate that CPEB phosphorylation and translation are regulated by CaMKII activity and provide a possible mechanism for how dendritic protein synthesis in the hippocampus may be stimulated during synaptic plasticity.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) has been implicated in the regulation of CRE-dependent transcription. To investigate the role of this kinase in neuronal plasticity and memory, we generated transgenic mice in which the expression of a dominant-negative form of CaMKIV (dnCaMKIV) is restricted to the postnatal forebrain. In these transgenic mice, activity-induced CREB phosphorylation and c-Fos expression were significantly attenuated. Hippocampal late LTP (L-LTP) was also impaired, whereas basic synaptic function and early LTP (E-LTP) were unaffected. These deficits correlated with impairments in long-term memory, specifically in its consolidation/retention phase but not in the acquisition phase. These results indicate that neural activity-dependent CaMKIV signaling in the neuronal nucleus plays an important role in the consolidation/retention of hippocampus-dependent long-term memory.

Although much has been learned about the neurobiological mechanisms underlying Pavlovian fear conditioning at the systems and cellular levels, relatively little is known about the molecular mechanisms underlying fear memory consolidation. The present experiments evaluated the role of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling cascade in the amygdala during Pavlovian fear conditioning. We first show that ERK/MAPK is transiently activated-phosphorylated in the amygdala, specifically the lateral nucleus (LA), at 60 min, but not 15, 30, or 180 min, after conditioning, and that this activation is attributable to paired presentations of tone and shock rather than to nonassociative auditory stimulation, foot shock sensitization, or unpaired tone-shock presentations. We next show that infusions of U0126, an inhibitor of ERK/MAPK activation, aimed at the LA, dose-dependently impair long-term memory of Pavlovian fear conditioning but leaves short-term memory intact. Finally, we show that bath application of U0126 impairs long-term potentiation in the LA in vitro. Collectively, these results demonstrate that ERK/MAPK activation is necessary for both memory consolidation of Pavlovian fear conditioning and synaptic plasticity in the amygdala.

The protein kinase C family of enzymes has been implicated in synaptic plasticity and memory in a wide range of animal species, but to date little information has been available concerning specific roles for individual isoforms of this category of kinases. To investigate the role of the beta isoform of PKC in mammalian learning, we characterized mice deficient in the PKC beta gene using anatomical, biochemical, physiological, and behavioral approaches. In our studies we observed that PKC beta was predominantly expressed in the neocortex, in area CA1 of the hippocampus, and in the basolateral nucleus of the amygdala. Mice deficient in PKC beta showed normal brain anatomy and normal hippocampal synaptic transmission, paired pulse facilitation, and long-term potentiation and normal sensory and motor responses. The PKC beta knock-out animals exhibited a loss of learning, however; they suffered deficits in both cued and contextual fear conditioning. The PKC expression pattern and behavioral phenotype in the PKC beta knock-out animals indicate a critical role for the beta isoform of PKC in learning-related signal transduction mechanisms, potentially in the basolateral nucleus of the amygdala.

Myelin basic protein (MBP) phosphorylation is a complex regulatory process that modulates the contribution of MBP to the stability of the myelin sheath. Recent research has demonstrated the modulation of MBP phosphorylation by mitogen-activated protein kinase (MAPK) during myelinogenesis and in the demyelinating disease multiple sclerosis. Here we investigated the physiological regulation of MBP phosphorylation by MAPK during neuronal activity in the alveus, the myelinated output fibers of the hippocampus. Using a phosphospecific antibody that recognizes the predominant MAPK phosphorylation site in MBP, Thr95, we found that MBP phosphorylation is regulated by high-frequency stimulation but not low-frequency stimulation of the alveus. This change was blocked by application of tetrodotoxin, indicating that action potential propagation in axons is required. It is interesting that the change in MBP phosphorylation was attenuated by the reactive oxygen species scavengers superoxide dismutase and catalase and the nitric oxide synthase inhibitor N-nitro-L-arginine. Removal of extracellular calcium also blocked the changes in MBP phosphorylation. Thus, we propose that during periods of increased neuronal activity, calcium activates axonal nitric oxide synthase, which generates the intercellular messengers nitric oxide and superoxide and regulates the phosphorylation state of MBP by MAPK.

Nonsynaptic signaling is becoming increasingly appreciated in studies of activity-dependent changes in the nervous system. We investigated the types of neuronal activity that elicit nonsynaptic communication between neurons and glial cells in hippocampal output fibers. High-frequency, but not low-frequency, action potential firing in myelinated CA1 axons of the hippocampus resulted in increased phosphorylation of the oligodendrocyte-specific protein myelin basic protein (MBP). This change was blocked by tetrodotoxin, indicating that axonally generated action potentials were necessary to regulate the phosphorylation state of MBP. Furthermore, scavengers of the reactive oxygen species superoxide and hydrogen peroxide and nitric oxide synthase inhibitors prevented activation of this neuron-glia signaling pathway. These results indicate that, during periods of increased neuronal activity in area CA1 of the hippocampus, reactive oxygen and nitrogen species are generated, which diffuse to neighboring oligodendrocytes and result in post-translational modifications of MBP, a key structural protein in myelin. Thus, in addition to their well-known capacity for activity-dependent neuron-neuron signaling, hippocampal pyramidal neurons possess a mechanism for activity-dependent neuron-glia signaling.

Although the biochemical mechanisms underlying learning and memory have not yet been fully elucidated, mounting evidence suggests that activation of protein kinases and phosphorylation of their downstream effectors plays a major role. Recent findings in our laboratory have shown a requirement for the mitogen-activated protein kinase (MAPK) cascade in hippocampal synaptic plasticity. Therefore, we used an inhibitor of MAPK activation, SL327, to test the role of the MAPK cascade in hippocampus-dependent learning in mice. SL327, which crosses the blood-brain barrier, was administered intraperitoneally at several concentrations to animals prior to cue and contextual fear conditioning. Administration of SL327 completely blocked contextual fear conditioning and significantly attenuated cue learning when measured 24 hr after training. To determine whether MAPK activation is required for spatial learning, we administered SL327 to mice prior to training in the Morris water maze. Animals treated with SL327 exhibited significant attenuation of water maze learning; they took significantly longer to find a hidden platform compared with vehicle-treated controls and also failed to use a selective search strategy during subsequent probe trials in which the platform was removed. These impairments cannot be attributed to nonspecific effects of the drug during the training phase; no deficit was seen in the visible platform task, and injection of SL327 following training produced no effect on the performance of these mice in the hidden platform task. These findings indicate that the MAPK cascade is required for spatial and contextual learning in mice.

Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

Mitogen-activated protein kinase (MAPK) is an integral component of cellular signaling during mitogenesis and differentiation of mitotic cells. Recently MAPK activation in post-mitotic cells has been implicated in hippocampal long-term potentiation (LTP), a potential cellular mechanism of learning and memory. Here we investigate the involvement of MAPK in learning and memory in behaving animals. MAPK activation increased in the rat hippocampus after an associative learning task, contextual fear conditioning. Two other protein kinases known to be activated during hippocampal LTP, protein kinase C and alpha-calcium/calmodulin protein kinase II, also were activated in the hippocampus after learning. Inhibition of the specific upstream activator of MAPK, MAPK kinase (MEK), blocked fear conditioning. Thus, classical conditioning in mammals activates MAPK, which is necessary for consolidation of the resultant learning.

The E6-AP ubiquitin ligase (human/mouse gene UBE3A/Ube3a) promotes the degradation of p53 in association with papilloma E6 protein, and maternal deficiency causes human Angelman syndrome (AS). Ube3a is imprinted with silencing of the paternal allele in hippocampus and cerebellum in mice. We found that the phenotype of mice with maternal deficiency (m-/p+) for Ube3a resembles human AS with motor dysfunction, inducible seizures, and a context-dependent learning deficit. Long-term potentiation (LTP) was severely impaired in m-/p+ mice despite normal baseline synaptic transmission and neuroanatomy, indicating that ubiquitination may play a role in mammalian LTP and that LTP may be abnormal in AS. The cytoplasmic abundance of p53 was increased in postmitotic neurons in m-/p+ mice and in AS, providing a potential biochemical basis for the phenotype through failure to ubiquitinate and degrade various effectors.

One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the alpha and betaII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca2+, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a "protected site" model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.

Hippocampal long-term potentiation (LTP) is a long-lasting and rapidly induced increase in synaptic strength. Previous experiments have determined that persistent activation of protein kinase C (PKC) contributes to the early maintenance phase of LTP (E-LTP). Using the back-phosphorylation method, we observed an increase in the phosphorylation of a 21-kDa PKC substrate, termed p21, 45 min after LTP was induced in the CA1 region of the hippocampus. p21 was found to have the same apparent molecular weight as the 18.5-kDa isoform of myelin basic protein (MBP) and was recognized by an antibody to MBP in western blotting and immunoprecipitation. Furthermore, p21 from control and potentiated hippocampal slices and purified MBP have identical phosphopeptide maps when back-phosphorylated and then digested with either endoproteinase Lys-C or endoproteinase Asp-N, suggesting that p21 and MBP are identical proteins. As there was no observed change in the amount of MBP in LTP, the increase in MBP phosphorylation during LTP cannot be explained by a change in the amount of protein. From these experiments, we conclude that the phosphorylation of the 18.5-kDa isoform of MBP is increased during E-LTP.