Mammalian target of rapamycin complex 1 activation negatively regulates Polo-like kinase 2-mediated homeostatic compensation following neonatal seizures.

Homeostatic plasticity is characterized by compensatory changes in synaptic strength and intrinsic membrane properties in response to chronic changes in neuronal activity. Neonatal seizures are a naturally occurring source of neuronal overactivation and can lead to long-term epilepsy and cognitive deficits. Using a rodent model of hypoxia-induced neonatal seizures that results in a persistent increase in AMPA receptor (AMPAR) function in hippocampal CA1 pyramidal neurons, we aimed to determine whether there was any evidence of an opposing endogenous homeostatic antiepileptic response. Given that this model results in long-term epilepsy, we also examined mechanisms whereby this homeostasis fails. Whole-cell patch-clamp recordings from neurons in slices removed at intervals following seizure onset revealed an initial up-regulation of AMPAR function that was followed by a transient dynamic attenuation of this enhancement by 48-72 h, although AMPAR function was still increased compared with nonseizure control baseline. This secondary down-regulation of enhanced AMPAR function was coincident with a marked transient increase in expression and function of the Polo-like kinase 2 (PLK2), which has previously been implicated in homeostatic down-regulation of neuronal excitability in cell/slice culture models. The effects were transient and at 1 wk AMPAR function once again became up-regulated, simultaneous with a decrease in PLK2 expression and function. This negative regulation was mediated by subacute postseizure increases in mammalian target of rapamycin (mTOR). Application of the mTOR inhibitor rapamycin prevented post-hypoxic seizure impairment of homeostasis, suggesting that homeostatic plasticity mechanisms may be potentially modifiable therapeutic targets in epileptogenesis.

Brain Perfusion Is Increased at Term in the White Matter of Very Preterm Newborns and Newborns with Congenital Heart Disease: Does this Reflect Activated Angiogenesis?

OBJECTIVE: This study aims to evaluate brain perfusion at term in very preterm newborns and newborns with congenital heart disease before their corrective surgery, and to search for histopathological indicators of whether the brain perfusion abnormalities of these newborns may be related to an activated angiogenesis. MATERIALS AND METHODS: Using magnetic resonance imaging and arterial spin labeling, regional cerebral blood flow was measured at a term-equivalent age for three very preterm newborns (born at < 32 weeks), one newborn with congenital heart disease before his corrective surgery and three healthy newborns. In addition, a histopathological analysis was performed on a newborn with congenital heart disease. RESULTS: The very preterm newborns and the newborn with congenital heart disease included in this study all displayed an increased signal in their white matter on T2-weighted imaging. The cerebral blood flow of these newborns was increased in their white matter, compared with the healthy term newborns. The vascular endothelial growth factor was overexpressed in the injured white matter of the newborn with congenital heart disease. CONCLUSION: Brain perfusion may be increased at term in the white matter, in very preterm newborns, and newborns with congenital heart disease, and it correlates with white matter abnormalities on conventional imaging.

α5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth.

Neural tumors often express neurotransmitter receptors as markers of their developmental lineage. Although these receptors have been well characterized in electrophysiological, developmental and pharmacological settings, their importance in the maintenance and progression of brain tumors and, importantly, the effect of their targeting in brain cancers remains obscure. Here, we demonstrate high levels of GABRA5, which encodes the α5-subunit of the GABAA receptor complex, in aggressive MYC-driven, "Group 3" medulloblastomas. We hypothesized that modulation of α5-GABAA receptors alters medulloblastoma cell survival and monitored biological and electrophysiological responses of GABRA5-expressing medulloblastoma cells upon pharmacological targeting of the GABAA receptor. While antagonists, inverse agonists and non-specific positive allosteric modulators had limited effects on medulloblastoma cells, a highly specific and potent α5-GABAA receptor agonist, QHii066, resulted in marked membrane depolarization and a significant decrease in cell survival. This effect was GABRA5 dependent and mediated through the induction of apoptosis as well as accumulation of cells in S and G2 phases of the cell cycle. Chemical genomic profiling of QHii066-treated medulloblastoma cells confirmed inhibition of MYC-related transcriptional activity and revealed an enrichment of HOXA5 target gene expression. siRNA-mediated knockdown of HOXA5 markedly blunted the response of medulloblastoma cells to QHii066. Furthermore, QHii066 sensitized GABRA5 positive medulloblastoma cells to radiation and chemotherapy consistent with the role of HOXA5 in directly regulating p53 expression and inducing apoptosis. Thus, our results provide novel insights into the synthetic lethal nature of α5-GABAA receptor activation in MYC-driven/Group 3 medulloblastomas and propose its targeting as a novel strategy for the management of this highly aggressive tumor.

Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia.

OBJECTIVE: The most common neurological symptom of tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) is early life refractory epilepsy. As previous studies have shown enhanced excitatory glutamatergic neurotransmission in TSC and FCD brains, we hypothesized that neurons associated with these lesions may also express altered γ-aminobutyric acid (GABA)(A) receptor (GABA(A)R)-mediated inhibition. METHODS: Expression of the GABA(A)R subunits α1 and α4, and the Na(+)-K(+)-2Cl(-) (NKCC1) and the K(+)-Cl(-) (KCC2) transporters, in human TSC and FCD type II specimens were analyzed by Western blot and double label immunocytochemistry. GABA(A) R responses in dysplastic neurons from a single case of TSC were measured by perforated patch recording and compared to normal-appearing cortical neurons from a non-TSC epilepsy case. RESULTS: TSC and FCD type IIb lesions demonstrated decreased expression of GABA(A)R α1, and increased NKCC1 and decreased KCC2 levels. In contrast, FCD type IIa lesions showed decreased α4, and increased expression of both NKCC1 and KCC2 transporters. Patch clamp recordings from dysplastic neurons in acute slices from TSC tubers demonstrated excitatory GABA(A)R responses that were significantly attenuated by the NKCC1 inhibitor bumetanide, in contrast to hyperpolarizing GABA(A)R-mediated currents in normal neurons from non-TSC cortical slices. INTERPRETATION: Expression and function of GABA(A)Rs in TSC and FCD type IIb suggest the relative benzodiazepine insensitivity and more excitatory action of GABA compared to FCD type IIa. These factors may contribute to resistance of seizure activity to anticonvulsants that increase GABAergic function, and may justify add-on trials of the NKCC1 inhibitor bumetanide for the treatment of TSC and FCD type IIb-related epilepsy.

Antiepileptic effects of levetiracetam in a rodent neonatal seizure model.

BACKGROUND: Neonatal seizures can result in chronic epilepsy and long-term behavioral and cognitive deficits. Levetiracetam (LEV), an antiepileptic drug that binds to the synaptic vesicle protein 2A (SV2A), has been increasingly used off-label for the therapy of neonatal seizures. Preclinical data regarding the acute or long-term efficacy of LEV are lacking. METHODS: We tested the anticonvulsant efficacy of LEV in a rat model of hypoxia-induced neonatal seizures. In addition, we evaluated the protective effects of postnatal day (P)10 LEV treatment on later-life kainic acid (KA)-induced seizure susceptibility and seizure-induced neuronal injury. Western blot and immunohistochemistry were used to assess the developmental regulation of SV2A in the rat and human brain. RESULTS: LEV pretreatment at P10 significantly decreased the cumulative duration of behavioral and electrographic seizures at both 25 and 50 mg/kg. At P40, KA-induced seizures and neuronal loss were significantly diminished in rats previously treated with LEV. LEV target SV2A is present in both neonatal rat and human brain and increases steadily to adulthood. CONCLUSION: LEV suppressed acute seizures induced by perinatal hypoxia and diminished later-life seizure susceptibility and seizure-induced neuronal injury, providing evidence for disease modification. These results support consideration of a clinical trial of LEV in neonatal seizures.

Modulation of lipid peroxidation and mitochondrial function improves neuropathology in Huntington's disease mice.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. Oxidative damage has been associated with pathological neuronal loss in HD. The therapeutic modulation of oxidative stress and mitochondrial function using low molecular weight compounds may be an important strategy for delaying the onset and slowing the progression of HD. In the present study, we found a marked increase of 4-hydroxy-2-nonenal (4-HNE) adducts, a lipid peroxidation marker, in the caudate and putamen of HD brains and in the striatum of HD mice. Notably, 4-HNE immunoreactivity was colocalized with mutant huntingtin inclusions in the striatal neurons of R6/2 HD mice. Administration of nordihydroguaiaretic acid (NDGA), an antioxidant that functions by inhibiting lipid peroxidation, markedly reduced 4-HNE adduct formation in the nuclear inclusions of R6/2 striatal neurons. NDGA also protected cultured neurons against oxidative stress-induced cell death by improving ATP generation and mitochondrial morphology and function. In addition, NDGA restored mitochondrial membrane potential, mitochondrial structure, and synapse structure in the striatum of R6/2 mice and increased their lifespan. The present findings suggest that further therapeutic studies using NDGA are warranted in HD and other neurodegenerative diseases characterized by increased oxidative stress and altered mitochondrial function.

Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis.

Toll receptors in Drosophila melanogaster function in morphogenesis and host defense. Mammalian orthologues of Toll, the Toll-like receptors (TLRs), have been studied extensively for their essential functions in controlling innate and adaptive immune responses. We report that TLR8 is dynamically expressed during mouse brain development and localizes to neurons and axons. Agonist stimulation of TLR8 in cultured cortical neurons causes inhibition of neurite outgrowth and induces apoptosis in a dissociable manner. Our evidence indicates that such TLR8-mediated neuronal responses do not involve the canonical TLR-NF-kappaB signaling pathway. These findings reveal novel functions for TLR8 in the mammalian nervous system that are distinct from the classical role of TLRs in immunity.

Development of later life spontaneous seizures in a rodent model of hypoxia-induced neonatal seizures.

PURPOSE: To study the development of epilepsy following hypoxia-induced neonatal seizures in Long-Evans rats and to establish the presence of spontaneous seizures in this model of early life seizures. METHODS: Long-Evans rat pups were subjected to hypoxia-induced neonatal seizures at postnatal day 10 (P10). Epidural cortical electroencephalography (EEG) and hippocampal depth electrodes were used to detect the presence of seizures in later adulthood (> P60). In addition, subdermal wire electrode recordings were used to monitor age at onset and progression of seizures in the juvenile period, at intervals between P10 and P60. Timm staining was performed to evaluate mossy fiber sprouting in the hippocampi of P100 adult rats that had experienced neonatal seizures. KEY FINDINGS: In recordings made from adult rats (P60-180), the prevalence of epilepsy in cortical and hippocampal EEG recordings was 94.4% following early life hypoxic seizures. These spontaneous seizures were identified by characteristic spike and wave activity on EEG accompanied by behavioral arrest and facial automatisms (electroclinical seizures). Phenobarbital injection transiently abolished spontaneous seizures. EEG in the juvenile period (P10-60) showed that spontaneous seizures first occurred approximately 2 weeks after the initial episode of hypoxic seizures. Following this period, spontaneous seizure frequency and duration increased progressively with time. Furthermore, significantly increased sprouting of mossy fibers was observed in the CA3 pyramidal cell layer of the hippocampus in adult animals following hypoxia-induced neonatal seizures. Notably, Fluoro-Jade B staining confirmed that hypoxic seizures at P10 did not induce acute neuronal death. SIGNIFICANCE: The rodent model of hypoxia-induced neonatal seizures leads to the development of epilepsy in later life, accompanied by increased mossy fiber sprouting. In addition, this model appears to exhibit a seizure-free latent period, following which there is a progressive increase in the frequency of electroclinical seizures.

T lymphocytes potentiate endogenous neuroprotective inflammation in a mouse model of ALS.

Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, progressive, motor neuron degenerative disease, in which the role of inflammation is not well established. Innate and adaptive immunity were investigated in the CNS of the Superoxide Dismutase 1 (SOD1)(G93A) transgenic mouse model of ALS. CD4+ and CD8+ T cells infiltrated SOD1(G93A) spinal cords during disease progression. Cell-specific flow cytometry and gene expression profiling showed significant phenotypic changes in microglia, including dendritic cell receptor acquisition, and expression of genes linked to neuroprotection, cholesterol metabolism and tissue remodeling. Microglia dramatically up-regulated IGF-1 and down-regulated IL-6 expression. When mutant SOD1 mice were bred onto a TCRbeta deficient background, disease progression was significantly accelerated at the symptomatic stage. In addition, microglia reactivity and IGF-1 levels were reduced in spinal cords of SOD1(G93A) (TCRbeta-/-) mice. These results indicate that T cells play an endogenous neuroprotective role in ALS by modulating a beneficial inflammatory response to neuronal injury.

Toll-like receptor 3 is a potent negative regulator of axonal growth in mammals.

Toll is a cell surface receptor with well described roles in the developmental patterning of invertebrates and innate immunity in adult Drosophila. Mammalian toll-like receptors represent a family of Toll orthologs that function in innate immunity by recognizing molecular motifs unique to pathogens or injured tissue. One member in this family of pattern recognition receptors, toll-like receptor 3 (TLR3), recognizes viral double-stranded RNA and host mRNA. We examined the expression and function of TLRs in the nervous system and found that TLR3 is expressed in the mouse central and peripheral nervous systems and is concentrated in the growth cones of neurons. Activation of TLR3 by the synthetic ligand polyinosine:polycytidylic acid (poly I:C) or by mRNA rapidly causes growth cone collapse and irreversibly inhibits neurite extension independent of nuclear factor kappaB. Mice lacking functional TLR3 were resistant to the neurodegenerative effects of poly I:C. Neonatal mice injected with poly I:C were found to have fewer axons exiting dorsal root ganglia and displayed related sensorimotor deficits. No effect of poly I:C was observed in mice lacking functional TLR3. Together, these findings provide evidence that an innate immune pattern recognition receptor functions autonomously in neurons to regulate axonal growth and advances a novel hypothesis that this class of receptors may contribute to injury and limited CNS regeneration.

WAVE1 is required for oligodendrocyte morphogenesis and normal CNS myelination.

Myelin formation involves the outgrowth of an oligodendrocyte cell process that can be regarded as a giant lamellipodium because it is an actively growing structure with extruded cytoplasm. The actin cytoskeleton is critical to morphogenesis, but little is known about regulation of actin dynamics in oligodendrocytes. Wiskott-Aldrich syndrome protein family verprolin homologous (WAVE) proteins mediate lamellipodia formation; thus, we asked whether these proteins function in oligodendrocyte process formation and myelination. Here, we show that WAVE1 is expressed by oligodendrocytes and localizes to the lamella leading edge where actin polymerization is actively regulated. CNS WAVE1 expression increases at the onset of myelination. Expression of dominant-negative WAVE1 impaired process outgrowth and lamellipodia formation in cultured oligodendrocytes. Similarly, oligodendrocytes isolated from mice lacking WAVE1 had fewer processes compared with controls, whereas neurons and astrocytes exhibited normal morphology. In white matter of WAVE1-/- mice, we found regional hypomyelination in the corpus callosum and to a lesser extent in the optic nerve. In optic nerve from WAVE1-/- mice, there were fewer nodes of Ranvier but nodal morphology was normal, implicating a defect in myelin formation. Our in vitro findings support a developmentally dynamic and cell-autonomous role for WAVE1 in regulating process formation in oligodendrocytes. Additionally, WAVE1 function during CNS myelination appears to be linked to regional cues. Although its loss can be compensated for in many CNS regions, WAVE1 is clearly required for normal amounts of myelin to form in corpus callosum and optic nerve. Together, these data demonstrate a role for WAVE1 in oligodendrocyte morphogenesis and myelination.

Role of cyclooxygenase-2 induction by transcription factor Sp1 and Sp3 in neuronal oxidative and DNA damage response.

Cyclooxygenase-2 (COX-2) has been implicated in neuronal survival and death. However, the precise regulatory mechanisms involved in COX-2 function are unclear. In the present study we found that COX-2 is induced in response to glutathione depletion-induced oxidative stress in primary cortical neurons. Two proximal specific Sp1 and Sp3 binding sites are responsible for the COX-2 promoter activity under normal as well as oxidative stress conditions through enhanced Sp1 and Sp3 DNA binding activity. Site-directed mutagenesis confirmed that -268/-267 positions serve as specific Sp1 and Sp3 recognition sites under oxidative stress. Enforced expression of Sp1 and Sp3 using HSV vectors increased the promoter activity, transcription, and protein level of COX-2 in cortical neurons. The dominant negative form of Sp1 abrogated the oxidative stress-induced promoter activity and expression of COX-2. We also demonstrated that adenovirus-mediated COX-2 gene delivery protected neurons from DNA damage induced by oxidative, genotoxic, and excitotoxic stresses and by ischemic injury. Moreover, COX-2(-/-) cortical neurons were more susceptible to DNA damage-induced cell death. These results indicate that in primary neurons Sp1 and Sp3 play an essential role in the modulation of COX-2 transcription, which mediates neuronal homeostasis and survival by preventing DNA damage in response to neuronal stress.

Atm-, p53-, and Gadd45a-deficient mice show an increased frequency of homologous recombination at different stages during development.

Atm, p53, and Gadd45a form part of a DNA-damage cellular response pathway; the absence of any one of these components results in increased genomic instability. We conducted an in vivo examination of the frequency of spontaneous homologous recombination in Atm-, p53-, or Gadd45a-deficient mice. In the absence of p53, we observed the greatest increase in events, a lesser increase in the absence of Atm, and only a modest increase in the absence of Gadd45a. The striking observation was the difference in the time at which the spontaneous events occurred in atm and trp53 mutant mice. The frequency of homologous recombination in atm mutant mice was increased later during development. In contrast, p53 appears to have a role in suppressing homologous recombination early during development, when p53 is known to spontaneously promote p21 activity. The timing of the increased spontaneous recombination was similar in the Gadd45a- and p53-deficient mice. This temporal resolution suggests that Atm and p53 can act to maintain genomic integrity by different mechanisms in certain in vivo contexts.

Increased Brain Perfusion Persists over the First Month of Life in Term Asphyxiated Newborns Treated with Hypothermia: Does it Reflect Activated Angiogenesis?

Many asphyxiated newborns still develop brain injury despite hypothermia therapy. The development of brain injury in these newborns has been related partly to brain perfusion abnormalities. The purposes of this study were to assess brain hyperperfusion over the first month of life in term asphyxiated newborns and to search for some histopathological clues indicating whether this hyperperfusion may be related to activated angiogenesis following asphyxia. In this prospective cohort study, regional cerebral blood flow was measured in term asphyxiated newborns treated with hypothermia around day 10 of life and around 1 month of life using magnetic resonance imaging (MRI) and arterial spin labeling. A total of 32 MRI scans were obtained from 24 term newborns. Asphyxiated newborns treated with hypothermia displayed an increased cerebral blood flow in the injured brain areas around day 10 of life and up to 1 month of life. In addition, we looked at the histopathological clues in a human asphyxiated newborn and in a rat model of neonatal encephalopathy. Vascular endothelial growth factor (VEGF) was expressed in the injured brain of an asphyxiated newborn treated with hypothermia in the first days of life and of rat pups 24-48 h after the hypoxic-ischemic event, and the endothelial cell count increased in the injured cortex of the pups 7 and 11 days after hypoxia-ischemia. Our data showed that the hyperperfusion measured by imaging persisted in the injured areas up to 1 month of life and that angiogenesis was activated in the injured brain of asphyxiated newborns.

Perfusion Imaging of Focal Cortical Dysplasia Using Arterial Spin Labeling: Correlation With Histopathological Vascular Density.

Focal cortical dysplasia is the most common malformation of cortical development, causing intractable epilepsy. This study investigated the relationship between brain perfusion and microvessel density in 7 children with focal cortical dysplasia. The authors analyzed brain perfusion measurements obtained by magnetic resonance imaging of 2 of the children and the microvessel density of brain tissue specimens obtained by epilepsy surgery on all of the children. Brain perfusion was approximately 2 times higher in the area of focal cortical dysplasia compared to the contralateral side. The microvessel density was nearly double in the area of focal cortical dysplasia compared to the surrounding cortex that did not have morphological abnormalities. These findings suggest that hyperperfusion can be related to increased microvessel density in focal cortical dysplasia rather than only to seizures. Further investigations are needed to determine the relationship between brain perfusion, microvessel density, and seizure activity.

Sensory innervation of the calvarial bones of the mouse.

Migraine sufferers frequently testify that their headache feels as if the calvarial bones are deformed, crushed, or broken (Jakubowski et al. [2006] Pain 125:286-295). This has lead us to postulate that the calvarial bones are supplied by sensory fibers. We studied sensory innervation of the calvaria in coronal and horizontal sections of whole-head preparations of postnatal and adult mice, via immunostaining of peripherin (a marker of thinly myelinated and unmyelinated fibers) or calcitonin gene-related peptide (CGRP; a marker more typical of unmyelinated nerve fibers). In pups, we observed nerve bundles coursing between the galea aponeurotica and the periosteum, between the periosteum and the bone, and between the bone and the meninges; as well as fibers that run inside the diploë in different orientations. Some dural fibers issued collateral branches to the pia at the frontal part of the brain. In the adult calvaria, the highest concentration of peripherin- and CGRP-labeled fibers was found in sutures, where they appeared to emerge from the dura. Labeled fibers were also observed in emissary canals, bone marrow, and periosteum. In contrast to the case in pups, no labeled fibers were found in the diploë of the adult calvaria. Meningeal nerves that infiltrate the periosteum through the calvarial sutures may be positioned to mediate migraine headache triggered by pathophysiology of extracranial tissues, such as muscle tenderness and mild trauma to the skull. In view of the concentration of sensory fibers in the sutures, it may be useful to avoid drilling the sutures in patients undergoing craniotomies for a variety of neurosurgical procedures.

Myelin structure and composition of myelinated tissue in the African lungfish.

To analyze myelin structure and the composition of myelinated tissue in the African lungfish (Protopterus dolloi), we used a combination of ultrastructural and biochemical techniques. Electron microscopy showed typical multilamellar myelin: CNS sheaths abutted one another, and PNS sheaths were separated by endoneurial collagen. The radial component, prominent in CNS myelin of higher vertebrates, was suggested by the pattern of staining but was poorly organized. The lipid and myelin protein compositions of lungfish tissues more closely resembled those of teleost than those of higher vertebrates (frog, mouse). Of particular note, for example, lungfish glycolipids lacked hydroxy fatty acids. Native myelin periodicities from unfixed nerves were in the range of those for higher vertebrates rather than for teleost fish. Lungfish PNS myelin had wider inter-membrane spaces compared with other vertebrates, and lungfish CNS myelin had spaces that were closer in value to those in mammalian than to amphibian or teleost myelins. The membrane lipid bilayer was narrower in lungfish PNS myelin compared to other vertebrates, whereas in the CNS myelin the bilayer was in the typical range. Lungfish PNS myelin showed typical compaction and swelling responses to incubation in acidic or alkaline hypotonic saline. The CNS myelin, by contrast, did not compact in acidic saline but did swell in the alkaline solution. This lability was more similar to that for the higher vertebrates than for teleost.