Seizure-induced neuronal apoptosis is related to dysregulation of the RNA-edited GluR2 subunit in the developing mouse brain.

Ca2+-permeable AMPA receptors (AMPAR) which crucially modify maturational programs of the developing brain are involved in seizure-induced glutamate excitotoxicity and apoptosis. Regulatory effects on AMPAR subunit composition and RNA-editing in the developing brain and their significance as therapeutic targets are not well understood. Here, we analyzed acute effects of recurrent pilocarpine-induced neonatal seizures on age- and region-specific expression of AMPAR subunits and adenosine deaminases (ADAR) in the developing mouse brain (P10). After recurrent seizure activity and regeneration periods of 6-72 h cerebral mRNA levels of GluR (glutamate receptor subunit) 1, GluR2, GluR3, and GluR4 were unaffected compared to controls. However, ratio of GluR2 and GluR4 to pooled GluR1-4 mRNA concentration significantly decreased in seizure-exposed brains in comparison to controls. After a regeneration period of 24-72 h ADAR1 and ADAR2 mRNA expression was significantly lower in seizure-exposed brains than in those of controls. This was confirmed at the protein level in the hippocampal CA3 region. We observed a regionally increased apoptosis (TUNEL+ and CC3+ cells) in the hippocampus, parietal cortex and subventricular zone of seizure-exposed brains in comparison to controls. Together, present in vivo data demonstrate the maturational age-specific, functional role of RNA-edited GluR2 in seizure-induced excitotoxicity in the developing mouse brain. In response to recurrent seizure activity, we observed reduced expression of GluR2 and the GluR2 mRNA-editing enzymes ADAR1 and ADAR2 accompanied by increased apoptosis in a region-specific manner. Thus, AMPA receptor subtype-specific mRNA editing is assessed as a promising target of novel neuroprotective treatment strategies in consideration of age-related developmental mechanisms.

Differential regulation of angiogenesis in the developing mouse brain in response to exogenous activation of the hypoxia-inducible transcription factor system.

Angiogenesis due to hypoxic-ischemic (HI) injury represents a crucial compensatory mechanism of the developing brain that is mainly regulated by hypoxia-inducible transcription factors (HIF). Pharmacological stimulation of HIF is suggested as a neuroprotective option, however, studies of its effects on vascular development are limited. We analyzed the influence of the prolyl-4-hydroxylase inhibitor (PHI), FG-4497, and erythropoietin (rhEPO) on post-hypoxic angiogenesis (angiogenic growth factors, vessel structures) in the developing mouse brain (P7) assessed after a regeneration period of 72 h. Exposure to systemic hypoxia (8% O2, 6 h) was followed by treatment (i.p.) with rhEPO (2500/5000 IU/kg) at 0, 24 and 48 h or FG-4497 (60/100 mg/kg) compared to controls. In response to FG-4497 treatment cortical and hippocampal vessel area and branching were significantly increased compared to controls. This was associated with elevated ANGPT-2 as well as decreased ANGPT-1 and TIE-2 mRNA levels. In response to rhEPO, mildly increased angiogenesis was associated with elevated ANGPT-2 but also TIE-2 mRNA levels in comparison to controls. In conclusion, present data demonstrate a differential regulation of the angiopoietin/TIE-2 system in response to PHI and rhEPO in the post-hypoxic developing brain pointing to potential functional consequences for vascular regeneration and vessel development.

Histopathological proof of the pathogenicity of a rare GFAP mutation in a patient with flaccid paraparesis.

Infantile Alexander disease is a rare progressive leukodystrophy caused by autosomal dominant mutations in the (GFAP) gene typically presenting with psychomotor retardation, progressive macrocephaly and refractory epilepsy. Neuroradiological hallmarks are extensive white matter lesions with frontal preponderance as well as signal intensity changes of basal ganglia and medulla oblongata with variable contrast enhancement. Here, we report an atypical manifestation in a 21-month-old boy presenting with flaccid paraparesis and areflexia. Cognitive, visual as well as fine motor skills and muscular strength of the upper extremities were appropriate for age. Weight and height as well as head circumference were within normal range. Clinical or electroencephalographic signs of seizures were absent. Cranial MRI demonstrated bifrontal cystic tumorous lesions with partial contrast rims, as well as space-occupying focal lesions of the caudate nuclei. Spinal MRI revealed swelling of the lumbar and cervical spinal cord. CSF and blood chemistry showed normal results. Histopathology of a subcortical lesion showed large amounts of Rosenthal fibers and protein droplets characteristic of Alexander disease. Sequencing detected a heterozygous mutation of the GFAP gene (c.205G > A; p.(Glu69Lys)) that has been reported before as probably pathogenetic in another case of lower spinal involvement. This well documented case draws attention to atypical spinal manifestations of Alexander disease and gives histopathological proof of the pathogenetic role of a rare GFAP mutation with marked spinal involvement.

Compound heterozygous RYR1 mutations in a preterm with arthrogryposis multiplex congenita and prenatal CNS bleeding.

RYR1 mutations, the most common cause of non-dystrophic neuromuscular disorders, are associated with the malignant hyperthermia susceptibility (MHS) trait as well as congenital myopathies with widely variable clinical and histopathological manifestations. Recently, bleeding anomalies have been reported in association with certain RYR1 mutations. Here we report a preterm infant born at 32 weeks gestation with arthrogryposis multiplex congenita due to compound heterozygous, previously MHS-associated RYR1 mutations, with additional signs of prenatal hemorrhage. The patient presented at birth with multiple joint contractures, scoliosis, severe thoracic rigidity and respiratory failure. He continued to depend on mechanical ventilation and tube feeding. Muscle histopathology showed a marked myopathic pattern with eccentric cores. Interestingly, the patient had additional unusual prenatal intraventricular hemorrhage, resulting in post-hemorrhagic hydrocephalus as well as epidural hemorrhage affecting the spinal cord. This report adds to the phenotypic variability associated with RYR1 mutations, and highlights possible bleeding complications in affected individuals.

Rare Variant of GM2 Gangliosidosis through Activator-Protein Deficiency.

GM2 gangliosidosis, AB variant, is a very rare form of GM2 gangliosidosis due to a deficiency of GM2 activator protein. We report on two patients with typical clinical features suggestive of GM2 gangliosidosis, but normal results for hexosaminidase A and hexosaminidase B as well as their corresponding genes. Genetic analysis of the gene encoding the activator protein, the GM2A gene, elucidated the cause of the disease, adding a novel mutation to the spectrum of GM2 AB variant. This report points out that in typical clinical constellations with normal enzyme results, genetic diagnostic for activator protein defects should be performed.

Hypoxia Potentiates LPS-Mediated Cytotoxicity of BV2 Microglial Cells In Vitro by Synergistic Effects on Glial Cytokine and Nitric Oxide System.

BACKGROUND: Microglial activation due to a variety of stimuli induces secretion of neurotoxic substances including inflammatory cytokines and nitric oxide (NO). Clinical studies indicate a cross-link between inflammatory and hypoxia-regulated pathways suggesting that bacterial infections markedly sensitize the immature brain to hypoxic injury. METHODS: The impact of inflammation and hypoxia on interleukin (IL)-1ß, IL-6, tumor necrosis factor α (TNF-α), and NO secretion and microglia-induced cytotoxicity was investigated exposing BV2 cells to lipopolysaccharides (LPS) and hypoxia (1% O2). Cytotoxicity, NO, and cytokine release was quantified by MTS and Griess assays and by enzyme-linked immunosorbent assays, respectively. RESULTS: LPS exposure of BV2 cells induced a significant, persistent production of NO, IL-1ß, IL-6, and TNF-α. Even after LPS removal, ongoing NO and cytokine secretion was observed. Hypoxia mediated exclusively a significant, short-term IL-1ß increase, but enhanced LPS-induced cytokine and NO secretion significantly. In addition, LPS-induced supernatants exhibited a stronger cytotoxic effect in glial and neuronal cells than LPS exposition (p < 0.001). Hypoxia potentiated LPS-induced cytotoxicity. CONCLUSION: Present data prove that LPS-induced soluble factors rather than LPS exposure mediate microglial toxicity under conditions of hypoxia in vitro. Apart from potential protective effects of the hypoxia-inducible transcription factor (HIF)-1α system, activation of proinflammatory pathways may markedly sensitize microglial cells to promote hypoxia-induced injuries of the developing brain.

Activin A in perinatal brain injury.

Activin A is a multifunctional growth and differentiation factor belonging to the transforming growth factor ß (TGF-ß) family. Growing evidence indicates its role as a neurotrophic factor and regulator of synaptic transmission as well as its functional importance in several types of cerebral injury. We recently described age-dependent expression of activin A and its regulation at the mRNA and protein level under different conditions of global hypoxia in the neonatal mouse brain. This review discusses the current knowledge of the function and regulation of activin A from human studies as well as from experimental models of brain injury focusing on acquired lesions of the developing rodent brain during the early stages of brain maturation.

Activin A regulation under global hypoxia in developing mouse brain.

Activin A is a multifunctional growth and differentiation factor with pronounced neuroprotective properties that is strongly up-regulated in various forms of acute brain disorders and injuries including epilepsy, stroke and trauma. In a pediatric context, activin A has been advanced as a potential marker for the severity of perinatal hypoxic-ischemic brain injury. Here we investigated the regulation of activin A under global hypoxia without ischemia in primary cultures of cortical neurons and in neonatal and adult mice of two strains (C57BL/6 and CD-1). From birth to adulthood, activin ßA subunit, activin receptors, and functional activin antagonists were all expressed at roughly similar mRNA levels in the brain of C57BL/6 mice. Independent of mouse line and age, we found both moderate (11% O2, 2h) and severe hypoxia (8%, 6h) to be consistently associated with normal or even reduced levels of activin ßA (Inhba) mRNA. The surprising unresponsiveness of Inhba expression to hypoxia was confirmed at the protein level. In situ hybridization did not indicate regional, hypoxia-related differences in Inhba expression. Pharmacologic stabilization of hypoxia inducible factors with the prolyl hydroxylase inhibitor FG-4497 did not influence Inhba mRNA levels in neonatal mice. Our data indicate that pure hypoxia differs from other, more complex types of brain damage in that it appears not to recruit activin A as an endogenous neuroprotective agent.

Ratiometric luminescence 2D in vivo imaging and monitoring of mouse skin oxygenation.

Tissue oxygenation plays a critical role in the pathogenesis of various diseases, but non-invasive, robust and user-friendly methods for its measurement in vivo still need to be established. Here, we are presenting an in vivo oxygen-detection system that uses ratiometric luminescence imaging (RLI) as a readout scheme to determine the skin oxygen tension of mouse hind footpads via side-by-side comparison with more established techniques including luminescence-lifetime imaging using planar sensor films and the polarographic electrode as the gold standard. We also demonstrate that this technology allows the detection of changes in mouse skin tissue oxygenation induced by subjecting mice to systemic hypoxia. The data demonstrate oxygen imaging based on RLI to be a most useful tool for reliably and easily analyzing and monitoring skin tissue oxygenation in vivo. This technology will advance our understanding of local regulation of skin tissue oxygenation in various disease conditions.

Novel MLL2 mutation in Kabuki syndrome with hypogammaglobulinemia and severe chronic thrombopenia.

BACKGROUND: Kabuki syndrome is a rare condition characterized by distinct dysmorphic features and a broad spectrum of organ anomalies. Differentiating it from other syndromes can be difficult, particularly in patients with incomplete phenotypic manifestation. Recently, MLL2 gene mutations were identified as the underlying genetic cause of Kabuki syndrome in the majority of cases. OBSERVATIONS: We report the case of an adolescent with an uncommon combination of manifestations, including hypogammaglobulinemia and severe chronic thrombopenia associated with a novel MLL2 mutation. CONCLUSIONS: This report adds to the growing knowledge on the mutational and phenotypic spectrum of Kabuki syndrome.

Classical MERRF phenotype associated with mitochondrial tRNA(Leu) (m.3243A>G) mutation.

Myoclonic epilepsy with ragged red fibres (MERRF) and mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) are established phenotypes of mitochondrial encephalopathies. Nearly all patients affected by MERRF harbour a mutation in the mitochondrial tRNA(Lys) gene. We report a 13-year-old patient who presented with the classical phenotype of MERRF but was found with the typical mutation of MELAS. The patient presented with myoclonic epilepsy beginning at 10 years of age, a muscle biopsy with ragged red fibres and some COX negative fibres and progressive bilateral MRI hyperintensitivities in the basal ganglia constituting MERRF syndrome but lacked clinical characteristics of MELAS. In particular, stroke-like episodes or lactic acidosis were not present. None of the tRNA mutations described in MERRF were found. However, further analyses showed the tRNA(Leu) mutation m.3243A>G usually found in MELAS to be responsible for the condition in this patient. This report highlights the broad phenotypic variability of mitochondrial encephalopathies with juvenile onset. It shows that m.3243A>G mutations can cause classical MERRF and emphasises the significance of comprehensive genetic studies if mitochondrial disease is suspected clinically.

Rapidly progressive phenotype of Lafora disease associated with a novel NHLRC1 mutation.

Lafora disease is a fatal, autosomal recessive form of progressive myoclonus epilepsy. Patients characteristically exhibit myoclonic and tonic-clonic seizures and cognitive impairment, beginning in their second decade. Alterations in two genes were identified as the cause of the disease. Mutations in the NHL repeat containing 1 (NHLRC1) gene were described in association with a more benign clinical course and later age of death, compared with epilepsy progressive myoclonus type 2A (EPM2A) mutations. We describe a rapidly progressive phenotype of Lafora disease in an adolescent patient with a novel NHLRC1 mutation. He developed severe disability and dementia less than 2 years after the onset of signs.