Fernando Pérez-Cerdá

The link between excitotoxic oligodendroglial death and demyelinating diseases

Oligodendrocytes, the myelinating cells of CNS axons, are highly vulnerable to excitotoxic signals mediated by glutamate receptors of the AMPA and kainate classes. Receptors in these cells are commonly activated by glutamate that is released from axons and glial cells. In addition, oligodendrocytes contribute to the control of extracellular glutamate levels by means of their own transporters. However, acute and chronic alterations in glutamate homeostasis can result in overactivation of AMPA and kainate receptors and subsequent excitotoxic oligodendroglial death. Furthermore, demyelinating lesions caused by excitotoxins can be similar to those observed in multiple sclerosis. This, together with the effect of AMPA and kainate receptor antagonists in ameliorating the neurological score of animals with experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis), indicates that oligodendrocyte excitotoxicity could be involved in the pathogenesis of demyelinating disorders.

P2X7 Receptor Blockade Prevents ATP Excitotoxicity in Oligodendrocytes and Ameliorates Experimental Autoimmune Encephalomyelitis

Oligodendrocyte death and demyelination are hallmarks of multiple sclerosis (MS). Here we show that ATP signaling can trigger oligodendrocyte excitotoxicity via activation of calcium-permeable P2X7 purinergic receptors expressed by these cells. Sustained activation of P2X7 receptors in vivo causes lesions that are reminiscent of the major features of MS plaques, i.e., demyelination, oligodendrocyte death, and axonal damage. In addition, treatment with P2X7 antagonists of chronic experimental autoimmune encephalomyelitis (EAE), a model of MS, reduces demyelination and ameliorates the associated neurological symptoms. Together, these results indicate that ATP can kill oligodendrocytes via P2X7 activation and that this cell death process contributes to EAE. Importantly, P2X7 expression is elevated in normal-appearing axon tracts in MS patients, suggesting that signaling through this receptor in oligodendrocytes may be enhanced in this disease. Thus, P2X7 receptor antagonists may be beneficial for the treatment of MS.

Excitotoxic damage to white matter.

Glutamate kills neurons by excitotoxicity, which is caused by sustained activation of glutamate receptors. In recent years, it has been shown that glutamate can also be toxic to white matter oligodendrocytes and to myelin by this mechanism. In particular, glutamate receptor‐mediated injury to these cells can be triggered by activation of alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid, kainate and N‐methyl‐d‐aspartate glutamate receptor types. Thus, these receptor classes, and the intermediaries of the signal cascades they activate, are potential targets for drug development to treat white matter damage in acute and chronic diseases. In addition, alterations of glutamate homeostasis in white matter can determine glutamate injury to oligodendrocytes and myelin. Astrocytes are responsible for most glutamate uptake in synaptic and non‐synaptic areas and consequently are the major regulators of glutamate homeostasis. Activated microglia in turn may secrete cytokines and generate radical oxygen species, which impair glutamate uptake and reduce the expression of glutamate transporters. Finally, oligodendrocytes also contribute to glutamate homeostasis. This review aims at summarizing the current knowledge about the mechanisms leading to oligodendrocyte cell death and demyelination as a consequence of alterations in glutamate signalling, and their clinical relevance to disease. In addition, we show evidence that oligodendrocytes can also be killed by ATP acting at P2X receptors. A thorough understanding of how oligodendrocytes and myelin are damaged by excitotoxicity will generate knowledge that can lead to improved therapeutic strategies to protect white matter.

Activation of Kainate Receptors Sensitizes Oligodendrocytes to Complement Attack

Glutamate excitotoxicity and complement attack have both been implicated separately in the generation of tissue damage in multiple sclerosis and in its animal model, experimental autoimmune encephalomyelitis. Here, we investigated whether glutamate receptor activation sensitizes oligodendrocytes to complement attack. We found that a brief incubation with glutamate followed by exposure to complement was lethal to oligodendrocytes in vitro and in freshly isolated optic nerves. Complement toxicity was induced by activation of kainate but not of AMPA receptors and was abolished by removing calcium from the medium during glutamate priming. Dose–response studies showed that sensitization to complement attack is induced by two distinct kainate receptor populations displaying high and low affinities for glutamate. Oligodendrocyte death by complement required the formation of the membrane attack complex, which in turn increased membrane conductance and induced calcium overload and mitochondrial depolarization as well as a rise in the level of reactive oxygen species. Treatment with the antioxidant Trolox and inhibition of poly(ADP-ribose) polymerase-1, but not of caspases, protected oligodendrocytes against damage induced by complement. These findings indicate that glutamate sensitization of oligodendrocytes to complement attack may contribute to white matter damage in acute and chronic neurological disorders.

Multiple sclerosis: novel perspectives on newly forming lesions

The mechanisms underlying lesion formation in multiple sclerosis are unknown. The prevailing view is that macrophages are primary mediators of myelin destruction in the relapsing and remitting forms of this disease. However, recent findings have revealed widespread oligodendrocyte apoptosis in the absence of a clear cellular immune response. These observations unveil a novel aspect of the pathogenesis of multiple sclerosis that is worthy of further exploration.

Functional glutamate transport in rodent optic nerve axons and glia

This is the author’s submitted draft of the paper published as Glia, 2009, 57 (11), pp. 1168-1177. The definitive version is available at www3.interscience.wiley.com, Doi: 10.1002/glia.20703.

Increased expression of glutamate transporters in subcortical white matter after transient focal cerebral ischemia.

Transient focal cerebral ischemia leads to extensive excitotoxic glial damage in the subcortical white matter. Efficient reuptake of released glutamate is essential for preventing glutamate receptor overstimulation and neuronal and glial death. The present study evaluates the expression of the main glutamate transporters (EAAT1, EAAT2, and EAAT3) in subcortical white matter of the rat after transient middle cerebral artery occlusion. Western blot analysis and immunohistochemistry show an increase in the expression of EAAT1 and EAAT2 in subcortical white matter early after ischemia which subsequently decreases at longer reperfusion periods. However, expression of both EAAT1 and EAAT2 remains higher in astrocytes forming the gliotic scar and in microglial/macrophage cells at the border of or within the infarct area, respectively. Taken together, these results indicate that there is a transient enhanced expression of EAATs in the subcortical white matter early after ischemia. Our findings reveal an adaptive response of subcortical white matter to increased levels of glutamate during focal cerebral ischemia which may limit excitotoxic damage.

KA1-like kainate receptor subunit immunoreactivity in neurons and glia using a novel anti-peptide antibody

Functional kainate receptors can be formed by various combinations of subunits with low (GluR5, GluR6 and GluR7) or high affinity (KA1 and KA2) for kainate. The precise contribution of each subunit to native receptors, as well as their distribution within the central nervous system (CNS) is still unclear. Here, we describe the presence of KA1-like immunoreactivity in both neurons and glial cells of the CNS, using a newly developed antiserum to a specific carboxy terminus epitope of the KA1 subunit. Intense immunoreactivity was observed in the CA3 area of the rat hippocampus. Electron microscopy revealed that immunostaining was present in dendritic structures postsynaptic to commissural-associational fibers, rather than in those contacted by mossy fiber terminals. We also observed immunostaining of CA1 pyramidal cell apical dendrites. In the cerebral cortex, KA1-like immunostaining was observed in many pyramidal neuron somata, mainly in layer V, and along their apical dendrites. A subset of gamma-amino-butyric acidic cells were also intensely stained. In the cerebellum, the antiserum selectively stained Purkinje cell somata and their dendrites as well as Bergmann glial processes. Other types of macroglia were also labeled by the KA1 antiserum. Thus, optic nerve oligodendrocytes both in vitro and in situ and cultured astrocytes were densely stained. Our results indicate that KA1-type subunits are more widely distributed throughout the CNS than previously thought. This newly developed antiserum may help to clarify the properties of kainate receptors containing KA1 or KA1-type subunits within the normal and pathological brain.

Localization of AMPA-selective glutamate receptor subunits in the adult cat visual cortex.

We have studied the presence and distribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-selective glutamate receptor subunits (GluR1, 2, 3, and 4) in the adult cat visual cortical areas 17, 18, 19, and the lateral suprasylvian areas (LSA). Reverse transcription-polymerase chain reaction (RT-PCR) amplification indicated that the genes encoding GluR1, 2, 3, and 4 are expressed in these areas and Western blot analysis revealed that the size of the corresponding peptides is similar to those described in the rat brain. In situ hybridization (ISH) using digoxigenin-labeled riboprobes showed that mRNAs coding for GluR1 and GluR3 were located in cells in all layers of the areas examined and also in the underlying white matter. GluR1 mRNA was relatively abundant throughout layers II-VI while GluR3 mRNA revealed a more laminated pattern of expression, preferentially labeling cells in layers II, III, V, and VI. The distribution of AMPA-selective receptor subunit peptides was studied by immunohistochemistry using subunit specific antibodies and found to be consistent with ISH results. In addition, we observed that most of the cells strongly labeled by the anti-GluR1 antibody were non-pyramidal neurons and that intense GluR2/3 immunoreactivity was seen preferentially in pyramidal neurons. Interestingly, double-labeling experiments indicated that neurons expressing gamma-aminobutyric acid (GABA) as well as the GluR1 subunit were particularly abundant in deeper layers. The GluR4 peptide was predominantly found in a relatively low number of layer III and layer V neurons with either pyramidal or non-pyramidal morphology. Finally, the distribution of neurons expressing the various receptor subunits was similar in all the visual cortical areas studied. These findings indicate a high expression of GluR1-3 subunits in the cat visual cortex and that GluR1 and GluR2/3 subunits are particularly abundant in non-pyramidal and pyramidal neurons, respectively. In addition, the results described here provide a reference for future studies dealing with the effect of visual deprivation on the expression of this receptor type.

Pío del Río Hortega and the discovery of the oligodendrocytes

Pío del Río Hortega (1882–1945) discovered microglia and oligodendrocytes (OLGs), and after Ramón y Cajal, was the most prominent figure of the Spanish school of neurology. He began his scientific career with Nicolás Achúcarro from whom he learned the use of metallic impregnation techniques suitable to study non-neuronal cells. Later on, he joined Cajal’s laboratory. and Subsequently, he created his own group, where he continued to develop other innovative modifications of silver staining methods that revolutionized the study of glial cells a century ago. He was also interested in neuropathology and became a leading authority on Central Nervous System (CNS) tumors. In parallel to this clinical activity, del Río Hortega rendered the first systematic description of a major polymorphism present in a subtype of macroglial cells that he named as oligodendroglia and later OLGs. He established their ectodermal origin and suggested that they built the myelin sheath of CNS axons, just as Schwann cells did in the periphery. Notably, he also suggested the trophic role of OLGs for neuronal functionality, an idea that has been substantiated in the last few years. Del Río Hortega became internationally recognized and established an important neurohistological school with outstanding pupils from Spain and abroad, which nearly disappeared after his exile due to the Spanish civil war. Yet, the difficulty of metal impregnation methods and their variability in results, delayed for some decades the confirmation of his great insights into oligodendrocyte biology until the development of electron microscopy and immunohistochemistry. This review aims at summarizing the pioneer and essential contributions of del Río Hortega to the current knowledge of oligodendrocyte structure and function, and to provide a hint of the scientific personality of this extraordinary and insufficiently recognized man.