Prenatal Alcohol Exposure and Chorioamnionitis Results in Microstructural Brain Injury in a Preclinical Investigation.

BACKGROUND: Prenatal Alcohol Exposure (PAE) impacts 2% to 5% of infants born in the United States yearly. Women who consume alcohol during pregnancy have a five-fold increased rate of Chorioamnionitis (CHORIO). Both PAE and CHORIO cause microstructural injury to multiple brain regions including major white matter tracts. OBJECTIVE: Utilizing two previously established animal models, we hypothesized that the combination of PAE+CHORIO would result in greater deficits in myelination and structural integrity than PAE alone. MATERIAL AND METHODS: Pregnant Long-Evans rats voluntarily drank 5% ethanol or saccharin until Gestational Day 19 (GD). On GD19, CHORIO was induced in one group of PAE dams by a 30 min uterine artery occlusion and injection of Lipopolysaccharide (LPS) into each amniotic sac. The remaining PAE dams and saccharin controls underwent sham surgery. Pups were born on GD22 and weaned on Postnatal Day 24 (PD). On PD28, offspring were sacrificed, and their brains examined using ex-vivo Diffusion Tensor Imaging (DTI). RESULTS: Compared to control, PAE alone did not affect offspring birth weights, mortality or any DTI measures. In contrast, PAE+CHORIO significantly reduced offspring survival and, in surviving pups, increased Radial Diffusivity (RD) in medial frontal cortex and decreased Fractional Anisotropy (FA) in medial and ventral frontal cortex and within capsular regions. CONCLUSION: The combination of moderate PAE+CHORIO results in an increased mortality, concomitant with diffuse microstructural brain injury noted in young adolescent offspring at PD28. Future studies should examine the extent to which PAE exacerbates the damage caused by CHORIO alone and whether these deficits persist into adulthood.

Prenatal Hypoxia-Ischemia Induces Abnormalities in CA3 Microstructure, Potassium Chloride Co-Transporter 2 Expression and Inhibitory Tone.

Infants who suffer perinatal brain injury, including those with encephalopathy of prematurity, are prone to chronic neurological deficits, including epilepsy, cognitive impairment, and behavioral problems, such as anxiety, inattention, and poor social interaction. These deficits, especially in combination, pose the greatest hindrance to these children becoming independent adults. Cerebral function depends on adequate development of essential inhibitory neural circuits and the appropriate amount of excitation and inhibition at specific stages of maturation. Early neuronal synaptic responses to γ-amino butyric acid (GABA) are initially excitatory. During the early postnatal period, GABAAR responses switch to inhibitory with the upregulation of potassium-chloride co-transporter KCC2. With extrusion of chloride by KCC2, the Cl(-) reversal potential shifts and GABA and glycine responses become inhibitory. We hypothesized that prenatal hypoxic-ischemic brain injury chronically impairs the developmental upregulation of KCC2 that is essential for cerebral circuit formation. Following late gestation hypoxia-ischemia (HI), diffusion tensor imaging in juvenile rats shows poor microstructural integrity in the hippocampal CA3 subfield, with reduced fractional anisotropy and elevated radial diffusivity. The loss of microstructure correlates with early reduced KCC2 expression on NeuN-positive pyramidal neurons, and decreased monomeric and oligomeric KCC2 protein expression in the CA3 subfield. Together with decreased inhibitory post-synaptic currents during a critical window of development, we document for the first time that prenatal transient systemic HI in rats impairs hippocampal CA3 inhibitory tone. Failure of timely development of inhibitory tone likely contributes to a lower seizure threshold and impaired cognitive function in children who suffer perinatal brain injury.

Kidins220/ARMS is expressed in neuroblastoma tumors and stabilizes neurotrophic signaling in a human neuroblastoma cell line.

BACKGROUND: Neurotrophic signaling is an important factor in the survival of developing neurons, and the expression of neurotrophic receptors correlates with prognosis in neuroblastoma. Kinase D-interacting substrate of 220 kDa (Kidins220) associates with neurotrophic receptors and stabilizes them, but the expression and function of Kidins220 in neuroblastoma are unknown. METHODS: We study Kidins220 expression in human neuroblastoma cell lines and tumor samples by western blotting and microarray analyses. We determine the functional consequences of downregulation of Kidins220 for response of cell lines to oxidative stress, chemotherapeutic treatment, and neurotrophins using small interfering RNA silencing and by measuring cell survival, signaling, and migration. RESULTS: Kidins220 is expressed in all neuroblastoma tumors and cell lines studied. Downregulation of Kidins220 leads to attenuation of nerve growth factor (NGF)-induced, but not brain-derived neurotrophic factor (BDNF)-induced, MAPK signaling. However, downregulation of Kidins220 does not alter the response to chemotherapeutic drugs or oxidative stress or affect cellular motility. CONCLUSION: Kidins220 is expressed in neuroblastoma tumors and stabilizes NGF-induced, but not BDNF-induced, survival signaling in neuroblastoma cell lines.

Kidins220/ARMS depletion is associated with the neural-to Schwann-like transition in a human neuroblastoma cell line model.

Peripheral neuroblastic tumors exist as a heterogeneous mixture of neuroblastic (N-type) cells and Schwannian stromal (S-type) cells. These stromal cells not only represent a differentiated and less aggressive fraction of the tumor, but also have properties that can influence the further differentiation of nearby malignant cells. In vitro neuroblastoma cultures exhibit similar heterogeneity with N-type and S-type cells representing the neuroblastic and stromal portions of the tumor, respectively, in behavior, morphology, and molecular expression patterns. In this study, we deplete kinase D-interacting substrate of 220kD (Kidins220) with an shRNA construct and thereby cause morphologic transition of the human SH-SY5Y neuroblastoma cell line from N-type to S-type. The resulting cells have similar morphology and expression profile to SH-EP1 cells, a native S-type cell line from the same parent cell line, and to SH-SY5Y cells treated with BrdU, a treatment that induces S-type morphology. Specifically, both Kidins220-deficient SH-SY5Y cells and native SH-EP1 cells demonstrate down-regulation of the genes DCX and STMN2, markers for the neuronal lineage. We further show that Kidins220, DCX and STMN2 are co-down-regulated in cells of S-type morphology generated by methods other than Kidins220 depletion. Finally, we report that the association of low Kidins220 expression with S-type morphology and low DCX and STMN2 expression is demonstrated in spontaneously occurring human peripheral neuroblastic tumors. We propose that Kidins220 is critical in N- to S-type transition of neural crest tumor cells.

p75NTR-dependent modulation of cellular handling of reactive oxygen species.

Our previous studies demonstrated that p75NTR confers protection against oxidative stress-induced apoptosis upon PC12 cells; however, the mechanisms responsible for this effect are not known. The present studies reveal decreased mitochondrion membrane potential and increased generation of reactive oxygen species (ROS) in p75NTR-deficient PC12 cells as well as diminution of ROS generation after transfection of a full-length p75NTR construct into these cells. They also show that p75NTR deficiency attenuates activation of the phosphatidylinositol 3-kinase --> phospho-Akt/protein kinase B pathway in PC12 cells by oxidative stress or neurotrophic ligands and inhibition of Akt phosphorylation decreases the glutathione (GSH) content in PC12 cells. In addition, decreased de novo GSH synthesis and increased GSH consumption are observed in p75NTR-deficient cells. These findings indicate that p75NTR regulates cellular handling of ROS to effect a survival response to oxidative stress.