Pamela L. Follett

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.

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.

Developmental regulation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. I. Rodent cerebral white matter and cortex

This is the first part of a two‐part study to investigate the cellular distribution and temporal regulation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid receptor (AMPAR) subunits in the developing white matter and cortex in rat (part I) and human (part II). Western blot and immunocytochemistry were used to evaluate the differential expression of AMPAR subunits on glial and neuronal subtypes during the first 3 postnatal weeks in the Long Evans and Sprague Dawley rat strains. In Long Evans rats during the first postnatal week, GluR2‐lacking AMPARs were expressed predominantly on white matter cells, including radial glia, premyelinating oligodendrocytes, and subplate neurons, whereas, during the second postnatal week, these AMPARs were highly expressed on cortical neurons, coincident with decreased expression on white matter cells. Immunocytochemical analysis revealed that cell‐specific developmental changes in AMPAR expression occurred 2–3 days earlier by chronological age in Sprague Dawley rats compared with Long Evans rats, despite overall similar temporal sequencing. In both white and gray matter, the periods of high GluR2 deficiency correspond to those of regional susceptibility to hypoxic/ischemic injury in each of the two rat strains, supporting prior studies suggesting a critical role for Ca2+‐permeable AMPARs in excitotoxic cellular injury and epileptogenesis. The developmental regulation of these receptor subunits strongly suggests that Ca2+ influx through GluR2‐lacking AMPARs may play an important role in neuronal and glial development and injury in the immature brain. Moreover, as demonstrated in part II, there are striking similarities between rat and human in the regional and temporal maturational regulation of neuronal and glial AMPAR expression. J. Comp. Neurol. 497:42–60, 2006.

Developmental regulation of AMPA receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury: Part II. Human cerebral white matter and cortex

This report is the second of a two‐part evaluation of developmental differences in α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid receptor (AMPAR) subunit expression in cell populations within white matter and cortex. In part I, we reported that, in rat, developmental expression of Ca2+‐permeable (GluR2‐lacking) AMPARs correlated at the regional and cellular level with increased susceptibility to hypoxia/ischemia (H/I), suggesting an age‐specific role of these receptors in the pathogenesis of brain injury. Part II examines the regional and cellular progression of AMPAR subunits in human white matter and cortex from midgestation through early childhood. Similarly to the case in the rodent, there is a direct correlation between selective vulnerability to H/I and expression of GluR2‐lacking AMPARs in human brain. For midgestational cases aged 20–24 postconceptional weeks (PCW) and for premature infants (25–37 PCW), we found that radial glia, premyelinating oligodendrocytes, and subplate neurons transiently expressed GluR2‐lacking AMPARs. Notably, prematurity represents a developmental window of selective vulnerability for white matter injury, such as periventricular leukomalacia (PVL). During term (38–42 PCW) and postterm neonatal (43–46 PCW) periods, age windows characterized by increased susceptibility to cortical injury and seizures, GluR2 expression was low in the neocortex, specifically on cortical pyramidal and nonpyramidal neurons. This study indicates that Ca2+‐permeable AMPAR blockade may represent an age‐specific therapeutic strategy for potential use in humans. Furthermore, these data help to validate specific rodent maturational stages as appropriate models for evaluation of H/I pathophysiology. J. Comp. Neurol. 497:61–77, 2006.

Mature myelin basic protein-expressing oligodendrocytes are insensitive to kainate toxicity

We examined the vulnerability to excitotoxicity of rat oligodendrocytes in dissociated cell culture at different developmental stages. Mature oligodendrocytes that express myelin basic protein were resistant to excitotoxic injury produced by kainate, whereas earlier stages in the oligodendrocyte lineage were vulnerable to this insult. To test the hypothesis that the sensitivity of immature oligodendrocytes and the resistance of mature oligodendrocytes to kainate toxicity were due to differences in membrane responsiveness to kainate, we used whole‐cell patch‐clamp recording. Oligodendrocyte precursors in cultures vulnerable to kainate toxicity responded to 500 μM kainate with large inward currents, whereas mature myelin basic protein‐expressing oligodendrocytes in cultures resistant to kainate toxicity showed no clear response to application of this agonist. We assayed expression of glutamate receptor subunits (GluR) ‐2, ‐4, ‐6, ‐7, and KA2 using immunoblot analysis and found that expression of all of these glutamate receptors was significantly down‐regulated in mature oligodendrocytes. These results suggest a striking developmental regulation of glutamate receptors in oligodendrocytes and suggest that the vulnerability of oligodendrocytes to non‐ N‐methyl‐D‐aspartate receptor‐mediated excitotoxicity might be much greater in developing oligodendrocytes than after the completion of myelination.

12-Lipoxygenase plays a key role in cell death caused by glutathione depletion and arachidonic acid in rat oligodendrocytes

Oxidative injury to premyelinating oligodendrocytes (preOLs) in developing white matter has been implicated in the pathogenesis of periventricular leukomalacia, the lesion underlying most cases of cerebral palsy in premature infants. In this study, we investigated the pathways of OL death induced by intracellular glutathione (GSH) depletion. We found that the lipoxygenase (LOX) inhibitors AA‐861 and BMD‐122 (N‐benzyl‐N‐hydroxy‐5‐phenylpentamide; BHPP), but not the cyclooxygenase (COX) inhibitor indomethacin, fully protected the cells from GSH depletion caused by cystine deprivation. Arachidonic acid (AA), the substrate for 12‐LOX, potentiated the toxicity of mild cystine deprivation and at higher concentration was itself toxic. This toxicity was also blocked by 12‐LOX inhibitors. Consistent with a role for 12‐LOX in the cell death pathway, 12‐LOX activity increased following cystine deprivation in OLs. Blocking 12‐LOX with AA‐861 effectively inhibited the accumulation of reactive oxygen species (ROS) induced by cystine deprivation. These data suggest that, in OLs, intracellular GSH depletion leads to activation of 12‐LOX, ROS accumulation and cell death. Mature OLs were more resistant than preOLs to cystine deprivation. The difference in sensitivity was not due to a difference in 12‐LOX activity but rather appeared to be related to the presence of stronger antioxidant defense mechanisms in mature OLs. These results suggest that 12‐LOX activation plays a key role in oxidative stress‐induced OL death.