Robert Fern

NMDA receptors are expressed in developing oligodendrocyte processes and mediate injury

Injury to oligodendrocyte processes, the structures responsible for myelination, is implicated in many forms of brain disorder. Here we show NMDA (N-methyl-d-aspartate) receptor subunit expression on oligodendrocyte processes, and the presence of NMDA receptor subunit messenger RNA in isolated white matter. NR1, NR2A, NR2B, NR2C, NR2D and NR3A subunits showed clustered expression in cell processes, but NR3B was absent. During modelled ischaemia, NMDA receptor activation resulted in rapid Ca2+-dependent detachment and disintegration of oligodendroglial processes in the white matter of mice expressing green fluorescent protein (GFP) specifically in oligodendrocytes (CNP-GFP mice). This effect occurred at mouse ages corresponding to both the initiation and the conclusion of myelination. NR1 subunits were found mainly in oligodendrocyte processes, whereas AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor subunits were mainly found in the somata. Consistent with this observation, injury to the somata was prevented by blocking AMPA/kainate receptors, and preventing injury to oligodendroglial processes required the blocking of NMDA receptors. The presence of NMDA receptors in oligodendrocyte processes explains why previous studies that have focused on the somata have not detected a role for NMDA receptors in oligodendrocyte injury. These NMDA receptors bestow a high sensitivity to acute injury and represent an important new target for drug development in a variety of brain disorders.

System xc− and Glutamate Transporter Inhibition Mediates Microglial Toxicity to Oligodendrocytes

Elevated levels of extracellular glutamate cause excitotoxic oligodendrocyte cell death and contribute to progressive oligodendrocyte loss and demyelination in white matter disorders such as multiple sclerosis and periventricular leukomalacia. However, the mechanism by which glutamate homeostasis is altered in such conditions remains elusive. We show here that microglial cells, in their activated state, compromise glutamate homeostasis in cultured oligodendrocytes. Both activated and resting microglial cells release glutamate by the cystine-glutamate antiporter system xc−. In addition, activated microglial cells act to block glutamate transporters in oligodendrocytes, leading to a net increase in extracellular glutamate and subsequent oligodendrocyte death. The blocking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors or the system xc− antiporter prevented the oligodendrocyte injury produced by exposure to LPS-activated microglial cells in mixed glial cultures. In a whole-mount rat optic nerve, LPS exposure produced wide-spread oligodendrocyte injury that was prevented by AMPA/kainate receptor block and greatly reduced by a system xc− antiporter block. The cell death was typified by swelling and disruption of mitochondria, a feature that was not found in closely associated axonal mitochondria. Our results reveal a novel mechanism by which reactive microglia can contribute to altering glutamate homeostasis and to the pathogenesis of white matter disorders.

Does astrocytic glycogen benefit axon function and survival in CNS white matter during glucose deprivation

Axons, the functional elements in CNS white matter, are frequently injured by ischemia, especially in the context of stroke. The pathophysiology of axonal injury induced by energy deprivation has been analyzed in the rat optic nerve and involves excessive calcium influx by way of reverse Na+/Ca2+ exchange and Ca2+ channels. Evidence is presented that CNS axonal function can be supported in the absence of glucose by intrinsic energy reserves provided through the breakdown of astrocytic glycogen. It is argued that energy is transferred from astrocytes to axons in the form of lactate, which is able to maintain axonal function when substituted for glucose. These observations complement the increasingly convincing hypothesis that astrocytes and neurons interact metabolically, both in the course of normal activity and under pathological conditions such as ischemia. The emerging picture would be no surprise to Camillo Golgi, who predicted a close facsimile of this glial‐neuronal interaction more than a century ago. GLIA 21:134–141, 1997.

Acute Lipopolysaccharide-Mediated Injury in Neonatal White Matter Glia: Role of TNF-α, IL-1β, and Calcium

Bacterial infection is implicated in the selective CNS white matter injury associated with cerebral palsy, a common birth disorder. Exposure to the bacterial endotoxin LPS produced death of white matter glial cells in isolated neonatal rat optic nerve (RON) (a model white matter tract), over a 180-min time course. A delayed intracellular Ca2+ concentration ([Ca2+]i) rise preceded cell death and both events were prevented by removing extracellular Ca2+. The cytokines TNF-α or IL-1β, but not IL-6, mimicked the cytotoxic effect of LPS, whereas blocking either TNF-α with a neutralizing Ab or IL-1 with recombinant antagonist prevented LPS cytotoxicity. Ultrastructural examination showed wide-scale oligodendroglial cell death in LPS-treated rat optic nerves, with preservation of astrocytes and axons. Fluorescently conjugated LPS revealed LPS binding on microglia and astrocytes in neonatal white and gray matter. Astrocyte binding predominated, and was particularly intense around blood vessels. LPS can therefore bind directly to developing white matter astrocytes and microglia to evoke rapid cell death in neighboring oligodendroglia via a calcium- and cytokine-mediated pathway. In addition to direct toxicity, LPS increased the degree of acute cell death evoked by ischemia in a calcium-dependent manner.

Hypoxia–ischemia preferentially triggers glutamate depletion from oligodendroglia and axons in perinatal cerebral white matter

Ischemia is implicated in periventricular white matter injury (PWMI), a lesion associated with cerebral palsy. PWMI features selective damage to early cells of the oligodendrocyte lineage, a phenomenon associated with glutamate receptor activation. We have investigated the distribution of glutamate in rat periventricular white matter at post-natal day 7. Immuno-electron microcopy was used to identify O4(+) oligodendroglia in control rats, and a similar approach was employed to stain glutamate in these cells before and after 90 mins of hypoxia-ischemia. This relatively brief period of hypoxia-ischemia produced mild cell injury, corresponding to the early stages of PWMI. Glutamate-like reactivity was higher in oligodendrocytes than in other cell types (2.13±0.25 counts/μm2), and declined significantly during hypoxia-ischemia (0.93±0.15 counts/μm2: P < 0.001). Astrocytes had lower glutamate levels (0.7±0.07 counts/μm2), and showed a relatively small decline during hypoxiaischemia. Axonal regions contained high levels of glutamate (1.84±0.20 counts/μm2), much of which was lost during hypoxia-ischemia (0.72±0.20 counts/μm2: P >0.001). These findings suggest that oligodendroglia and axons are the major source of extracellular glutamate in developing white matter during hypoxia-ischemia, and that astrocytes fail to accumulate the glutamate lost from these sources. We also examined glutamate levels in the choroid plexus. Control glutamate levels were high in both choroid epithelial (1.90±0.20 counts/μm2), and ependymal cells (2.20±0.28 counts/μm2), and hypoxia-ischemia produced a large fall in ependymal glutamate (0.97±0.08 counts/μm2: P >0.001). The ependymal cells were damaged by the insult and represent a further potential source of glutamate during ischemia.

White matter injury: Ischemic and nonischemic

Ischemic pathologies of white matter (WM) include a large proportion of stroke and developmental lesions while multiple sclerosis (MS) is the archetype nonischemic pathology. Growing evidence suggests other important diseases including neurodegenerative and psychiatric disorders also involve a significant WM component. Axonal, oligodendroglial, and astroglial damage proceed via distinct mechanisms in ischemic WM and these mechanisms evolve dramatically with maturation. Axons may pass through four developmental stages where the pattern of membrane protein expression influences how the structure responds to ischemia; WM astrocytes pass through at least two and differ significantly in their ischemia tolerance from grey matter astrocytes; oligodendroglia pass through at least three, with the highly ischemia intolerant pre‐oligodendrocyte (pre‐Oli) stage linking the less sensitive precursor and mature phenotypes. Neurotransmitters play a central role in WM pathology at all ages. Glutamate excitotoxicity in WM has both necrotic and apoptotic components; the latter mediated by intracellular pathways which differ between receptor types. ATP excitotoxicity may be largely mediated by the P2X7 receptor and also has both necrotic and apoptotic components. Interplay between microglia and other cell types is a critical element in the injury process. A growing appreciation of the significance of WM injury for nonischemic neurological disorders is currently stimulating research into mechanisms; with curious similarities being found with those operating during ischemia. A good example is traumatic brain injury, where axonal pathology can proceed via almost identical pathways to those described during acute ischemia. GLIA 2014;62:1780–1789

Glutamate receptor-mediated ischemic injury of premyelinated central axons†

Ischemic injury of axons is a feature of periventricular leukomalacia, a pathological correlate of cerebral palsy. Recent evidence suggests that axons are damaged before they receive the first layer of compact myelin. Here we examine the cellular mechanisms underlying ischemic‐type injury of premyelinated central axons.

Neurotransmitter Signaling in White Matter

White matter (WM) tracts are bundles of myelinated axons that provide for rapid communication throughout the CNS and integration in grey matter (GM). The main cells in myelinated tracts are oligodendrocytes and astrocytes, with small populations of microglia and oligodendrocyte precursor cells. The prominence of neurotransmitter signaling in WM, which largely exclude neuronal cell bodies, indicates it must have physiological functions other than neuron‐to‐neuron communication. A surprising aspect is the diversity of neurotransmitter signaling in WM, with evidence for glutamatergic, purinergic (ATP and adenosine), GABAergic, glycinergic, adrenergic, cholinergic, dopaminergic and serotonergic signaling, acting via a wide range of ionotropic and metabotropic receptors. Both axons and glia are potential sources of neurotransmitters and may express the respective receptors. The physiological functions of neurotransmitter signaling in WM are subject to debate, but glutamate and ATP‐mediated signaling have been shown to evoke Ca2+ signals in glia and modulate axonal conduction. Experimental findings support a model of neurotransmitters being released from axons during action potential propagation acting on glial receptors to regulate the homeostatic functions of astrocytes and myelination by oligodendrocytes. Astrocytes also release neurotransmitters, which act on axonal receptors to strengthen action potential propagation, maintaining signaling along potentially long axon tracts. The co‐existence of multiple neurotransmitters in WM tracts suggests they may have diverse functions that are important for information processing. Furthermore, the neurotransmitter signaling phenomena described in WM most likely apply to myelinated axons of the cerebral cortex and GM areas, where they are doubtless important for higher cognitive function. GLIA 2014;62:1762–1779

Vesicular apparatus, including functional calcium channels, are present in developing rodent optic nerve axons and are required for normal node of Ranvier formation

P/Q‐type calcium channels are known to form clusters at the presynaptic membrane where they mediate calcium influx, triggering vesicle fusion. We now report functional P/Q channel clusters in the axolemma of developing central axons that are also associated with sites of vesicle fusion. These channels were activated by axonal action potentials and the resulting calcium influx is well suited to mediate formation of a synaptic style SNARE complex involving SNAP‐25, that we show to be located on the axolemma. Vesicular elements within axons were found to be the sole repository of vesicular glutamate in developing white matter. The axonal vesicular elements expressed the glutamate transporter V‐ATPase, which is responsible for vesicular glutamate loading. The P/Q channel α1A subunit was found to be present within the axolemma at early nodes of Ranvier and deleterious mutations of the α1A subunit, or an associated α2δ‐2 subunit, disrupted the localization of nodal proteins such as voltage‐gated sodium channels, β IV spectrin and CASPR‐1. This was associated with the presence of malformed nodes of Ranvier characterized by an accumulation of axoplasmic vesicles under the nodal membrane. The data are consistent with the presence of a vesicular signalling pathway between axons and glial cells that is essential for proper development of the node of Ranvier.

GFP imaging of live astrocytes: regional differences in the effects of ischaemia upon astrocytes

The basic division between white matter ‘fibrous’ astrocytes and grey matter ‘protoplasmic’ astrocytes is well established in terms of their morphological differences. The availability of transgenic animals with green fluorescent protein (GFP) expression restricted to specific glial cell types now provides an approach for looking at changes in cell number and morphology in the two astrocyte types in whole mount preparations. This is an important goal, as the ease of generating astrocyte cultures has led to a proliferation of studies that have examined ischaemic effects on astrocytes in vitro. This has in turn engendered a belief that astrocytes have an extraordinary resistance to ischaemic injury, a belief that runs counter to almost all the data available from in vivo and whole‐mount preparations. One possible source of this confusion is the reactive changes that occur in astrocytes following injury, which include an increase in cell number that may obscure early astrocyte cell death and which has been reported to initiate within hours of an ischaemic event. However, we show here that neither white matter nor grey matter GFP(+) astrocytes exhibit any feature of reactive astrocytosis within a 180‐min period of reperfusion following modelled ischaemia in neonatal whole‐mount preparations. We also show that white matter astrocytes are much more sensitive to ischaemia‐reperfusion injury than are grey matter astrocytes, a feature that may have high significance for developmental disorders of white matter tracts such as cerebral palsy.