A negative allosteric modulator of PDE4D enhances learning after traumatic brain injury.

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.

Chronic Cognitive Dysfunction after Traumatic Brain Injury Is Improved with a Phosphodiesterase 4B Inhibitor.

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.

Phosphodiesterase inhibition rescues chronic cognitive deficits induced by traumatic brain injury.

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.

Enhancing cognitive function in chronic TBI: The Role of α7 nicotinic acetylcholine receptor modulation.

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.

Enhancing cognitive recovery in chronic traumatic brain injury through simultaneous allosteric modulation of α7 nicotinic acetylcholine and α5 GABAA receptors.

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 GABAA receptors (GABAARs), 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 GABAARs. 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 GABAAR signaling may be a potential therapy for treating cognitive deficits during chronic recovery from TBI.

Proinflammatory cytokine regulation of cyclic AMP-phosphodiesterase 4 signaling in microglia in vitro and following CNS injury.

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)-1ß 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-1ß stimulation produced a profound reduction (>90%) of cyclic AMP within EOC2 microglia from 30 min that then recovered after IL-1ß 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-1ß, 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-1ß 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-1ß. 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-1ß 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.

STAT3 signaling after traumatic brain injury.

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 ß, oncostatin M, matrix metalloproteinase 3, cyclin-dependent kinase inhibitor 1A, CCAAT/enhancer-binding protein ß, 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.

Phosphodiesterase isoform-specific expression induced by traumatic brain injury.

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.

Genetic ablation of metabotropic glutamate receptor 5 in rats results in an autism-like behavioral phenotype.

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.

Early Life Stress Exacerbates Outcome after Traumatic Brain Injury.

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.

Post-traumatic seizure susceptibility is attenuated by hypothermia therapy.

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.

EphB3 interacts with initiator caspases and FHL-2 to activate dependence receptor cell death in oligodendrocytes after brain injury.

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.

Glycogen synthase kinase-3 inhibition rescues sex-dependent contextual fear memory deficit in human immunodeficiency virus-1 transgenic mice.

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.

Positive allosteric modulation of the α7 nicotinic acetylcholine receptor as a treatment for cognitive deficits after traumatic brain injury.

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.

Activated c-Jun N-terminal kinase is required for axon formation.

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.

Alterations in mammalian target of rapamycin signaling pathways after traumatic brain injury.

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.

Is temperature an important variable in recovery after mild traumatic brain injury?

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.

Therapeutic benefits of phosphodiesterase 4B inhibition after traumatic brain injury.

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.

Bidirectional regulation of cytoplasmic polyadenylation element-binding protein phosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein phosphatase 1 during hippocampal long-term potentiation.

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.

Activation of calcium/calmodulin-dependent protein kinases after traumatic brain injury.

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.