Paul A. Rosenberg

Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway

Innate immunity is an evolutionarily ancient system that provides organisms with immediately available defense mechanisms through recognition of pathogen-associated molecular patterns. We show that in the CNS, specific activation of innate immunity through a Toll-like receptor 4 (TLR4)-dependent pathway leads to neurodegeneration. We identify microglia as the major lipopolysaccharide (LPS)-responsive cell in the CNS. TLR4 activation leads to extensive neuronal death in vitro that depends on the presence of microglia. LPS leads to dramatic neuronal loss in cultures prepared from wild-type mice but does not induce neuronal injury in CNS cultures derived from tlr4 mutant mice. In an in vivo model of neurodegeneration, stimulating the innate immune response with LPS converts a subthreshold hypoxic-ischemic insult from no discernable neuronal injury to severe axonal and neuronal loss. In contrast, animals bearing a loss-of-function mutation in the tlr4 gene are resistant to neuronal injury in the same model. The present study demonstrates a mechanistic link among innate immunity, TLRs, and neurodegeneration.

Hundred-fold increase in neuronal vulnerability to glutamate toxicity in astrocyte-poor cultures of rat cerebral cortex

In cultures of rat cerebral cortex in which astrocyte proliferation was stringently suppressed, glutamate neurotoxicity occurred at glutamate concentrations similar to those which are normally found in the extracellular space in the hippocampus. Concentrations of glutamate one hundred-fold higher were required to produce neurotoxicity in the presence of abundant astrocytes. This suggests that the sensitivity of central neurons to glutamate toxicity may be dependent upon astrocyte function.

Glutamate Receptor-Mediated Oligodendrocyte Toxicity in Periventricular Leukomalacia: A Protective Role for Topiramate

Periventricular leukomalacia is a form of hypoxic–ischemic cerebral white matter injury seen most commonly in premature infants and is the major antecedent of cerebral palsy. Glutamate receptor-mediated excitotoxicity is a predominant mechanism of hypoxic–ischemic injury to developing cerebral white matter. We have demonstrated previously the protective effect of AMPA–kainate-type glutamate receptor blockade in a rodent model of periventricular leukomalacia. The present study explores the therapeutic potential of glutamate receptor blockade for hypoxic–ischemic white matter injury. We demonstrate that AMPA receptors are expressed on developing human oligodendrocytes that populate fetal white matter at 23–32 weeks gestation, the period of highest risk for periventricular leukomalacia. We show that the clinically available anticonvulsant topiramate, when administered post-insult in vivo, is protective against selective hypoxic–ischemic white matter injury and decreases the subsequent neuromotor deficits. We further demonstrate that topiramate attenuates AMPA–kainate receptor-mediated cell death and calcium influx, as well as kainate-evoked currents in developing oligodendrocytes, similar to the AMPA–kainate receptor antagonist 6-nitro-7-sulfamoylbenzo-(f)quinoxaline-2,3-dione (NBQX). Notably, protective doses of NBQX and topiramate do not affect normal maturation and proliferation of oligodendrocytes either in vivo or in vitro. Taken together, these results suggest that AMPA–kainate receptor blockade may have potential for translation as a therapeutic strategy for periventricular leukomalacia and that the mechanism of protective efficacy of topiramate is caused at least in part by attenuation of excitotoxic injury to premyelinating oligodendrocytes in developing white matter.

Glutathione Peroxidase-Catalase Cooperativity Is Required for Resistance to Hydrogen Peroxide by Mature Rat Oligodendrocytes

Oxidative mechanisms of injury are important in many neurological disorders, including hypoxic-ischemic brain damage. Cerebral palsy after preterm birth is hypothesized to be caused by hypoxic-ischemic injury of developing oligodendrocytes (OLs). Here we examined the developmental sensitivity of OLs to exogenous hydrogen peroxide (H2O2) with stage-specific rat oligodendrocyte cultures. We found that H2O2 itself or that generated by glucose oxidase was more toxic to developing than to mature OLs. Mature OLs were able to degrade H2O2 faster than developing OLs, suggesting that higher antioxidant enzyme activity might be the basis for their resistance. Catalase expression and activity were relatively constant during oligodendrocyte maturation, whereas glutathione peroxidase (GPx) was upregulated with a twofold to threefold increase in its expression and activity. Thus, it appeared that the developmental change in resistance to H2O2 was caused by modulation of GPx but not by catalase expression. To test the relative roles of catalase and GPx in the setting of oxidative stress, we measured enzyme activity in cells exposed to H2O2 and found that H2O2 induced a decrease in catalase activity in developing but not in mature OLs. Inhibition of GPx by mercaptosuccinate led to an increase in the vulnerability of mature OLs to H2O2 as well as a reduction in catalase activity. Finally, H2O2-dependent inactivation of catalase in developing OLs was prevented by the GPx mimic ebselen. These data provide evidence for a key role for GPx-catalase cooperativity in the resistance of mature OLs to H2O2-induced cell death.

The developing oligodendrocyte: key cellular target in brain injury in the premature infant

Brain injury in the premature infant, a problem of enormous importance, is associated with a high risk of neurodevelopmental disability. The major type of injury involves cerebral white matter and the principal cellular target is the developing oligodendrocyte. The specific phase of the oligodendroglial lineage affected has been defined from study of both human brain and experimental models. This premyelinating cell (pre‐OL) is vulnerable because of a series of maturation‐dependent events. The pathogenesis of pre‐OL injury relates to operation of two upstream mechanisms, hypoxia‐ischemia and systemic infection/inflammation, both of which are common occurrences in premature infants. The focus of this review and of our research over the past 15–20 years has been the cellular and molecular bases for the maturation‐dependent vulnerability of the pre‐OL to the action of the two upstream mechanisms. Three downstream mechanisms have been identified, i.e., microglial activation, excitotoxicity and free radical attack. The work in both experimental models and human brain has identified a remarkable confluence of maturation‐dependent factors that render the pre‐OL so exquisitely vulnerable to these downstream mechanisms. Most importantly, elucidation of these factors has led to delineation of a series of potential therapeutic interventions, which in experimental models show marked protective properties. The critical next step, i.e., clinical trials in the living infant, is now on the horizon.

The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons

GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3′-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14–29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.

Cell and fiber type distribution of dystrophin

Duchenne muscular dystrophy is the result of dystrophin deficiency. We have determined the cell types likely to express the pathogenic effects of this neuromuscular disease by determining the pattern of dystrophin expression in normal cells. We find that all physiological types of muscle cells express dystrophin at similar levels, and that the dystrophin content of various tissues correlates with the myogenic cell population of each tissue. The dystrophin content of brain and spinal cord, however, is found not to correlate with any type of muscle cell, and it is suggested that neurons express dystrophin. The potential involvement of striated muscle fibers, the vasculature, and the nervous system in the etiology of Duchenne muscular dystrophy makes it likely that the disease is a complex disorder of combined pathogenesis. We also find that the dystrophic chicken does not represent an animal model for dystrophin deficiency.

Calcium-permeable AMPA/kainate receptors mediate toxicity and preconditioning by oxygen-glucose deprivation in oligodendrocyte precursors

Hypoxic–ischemic brain injury in premature infants results in cerebral white matter lesions with prominent oligodendroglial injury and loss, a disorder termed periventricular leukomalacia (PVL). We have previously shown that glutamate receptors mediate hypoxic–ischemic injury to oligodendroglial precursor cells (OPCs) in a model of PVL in the developing rodent brain. We used primary OPC cultures to examine the mechanism of cellular toxicity induced by oxygen–glucose deprivation (OGD) to simulate brain ischemia. OPCs were more sensitive to OGD-induced toxicity than mature oligodendrocytes, and OPC toxicity was attenuated by nonselective [2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX), 6-cyano-7-nitroquinoxaline-2,3-dione], α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring (GYKI 52466), kainate-preferring (γ-d-glutamylaminomethanesulfonic acid), or Ca2+-permeable AMPA/kainate receptor antagonists (joro spider toxin, JSTx) administered either during or after OGD. Furthermore, NBQX or JSTx blocked OGD-induced Ca2+ influx. Relevant to recurrent hypoxic–ischemic insults in developing white matter, we examined the effects of sublethal OGD preconditioning. A prior exposure of OPCs to sublethal OGD resulted in enhanced vulnerability to subsequent excitotoxic or OGD-induced injury associated with an increased Ca2+ influx. AMPA/kainate receptor blockade with NBQX or JSTx either during or after sublethal OGD prevented its priming effect. Furthermore, OGD preconditioning resulted in a down-regulation of the AMPA receptor subunit GluR2 on cell surface that increased Ca2+ permeability of the receptors. Overall, these data suggest that aberrantly enhanced activation of Ca2+-permeable AMPA/kainate receptors may be a major mechanism in acute and repeated hypoxic–ischemic injury to OPCs in disorders of developing cerebral white matter, such as PVL.

Responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures

AIM To report several types of response of immature permanent teeth with infected necrotic pulp tissue and either apical periodontitis or abscess to revascularization procedures. METHODOLOGY Twenty immature permanent teeth with infected necrotic pulp tissue and either apical periodontitis or abscesses from 20 patients were included. The teeth were isolated with rubber dam, and pulp chambers was accessed through the crowns. The canals were gently irrigated with 5.25% sodium hypochlorite with minimal mechanical debridement. Calcium hydroxide was used as an inter-appointment intracanal medicament and placed into the coronal half of the canal space. After resolution of clinical signs and symptoms, bleeding was induced into the canal space from the periapical tissues using K-files. The coronal canal space was sealed with a mixture of mineral trioxide aggregate (MTA) and saline solution. The access cavity was filled with composite resin. These immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscesses were followed up from 6 to 26 months. RESULTS Five types of responses of these immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures were observed: type 1, increased thickening of the canal walls and continued root maturation; type 2, no significant continuation of root development with the root apex becoming blunt and closed; type 3, continued root development with the apical foramen remaining open; type 4, severe calcification (obliteration) of the canal space; type 5, a hard tissue barrier formed in the canal between the coronal MTA plug and the root apex. CONCLUSIONS Based on this case series, the outcome of continued root development was not as predictable as increased thickening of the canal walls in human immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess after revascularization procedures. Continued root development of revascularized immature permanent necrotic teeth depends on whether the Hertwigs epithelial root sheath survives in case of apical periodontitis/abscess. Severe pulp canal calcification (obliteration) by hard tissue formation might be a complication of internal replacement resorption or union between the intracanal hard tissue and the apical bone (ankylosis) in revascularized immature permanent necrotic teeth.

Peroxynitrite-Induced Neuronal Apoptosis Is Mediated by Intracellular Zinc Release and 12-Lipoxygenase Activation

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study, we observed that TPEN [N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine], a zinc chelator, protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor, diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition, we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX), p38 mitogen-activated protein kinase (MAPK), and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX, generation of reactive oxygen species (ROS), and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX, ROS accumulation, p38 activation, and caspase-3 activation. Therefore, therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation, stroke, and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death.