Expression of glutamate transporter subtypes during normal human corticogenesis and type II lissencephaly

Abstract

Glutamate transporters are thought to have an important role in central nervous system (CNS) development. We investigated the expression of the sodium-dependent high-affinity glutamate transporters EAAT1, EAAT2, and EAAT3 in 11 human autopsied cases without neurological disorders and in four cases with type II lissencephaly including Walker Warburg's syndrome (WWS) and Fukuyama-type congenital muscular dystrophy (FCMD), both of which are classified as migration disorders of the human brain. Expression of glutamate transporter subtypes was differentially regulated during normal human corticogenesis. Although EAAT1 and EAAT2 were mainly localized to the cortical astrocytes in the postnatal brain, EAAT1 was enriched in the proliferative zones and radial glia from 13 gestational weeks (GW) to 20 GW. EAAT2 was abundant in the intermediate zone until 23 GW, and transiently expressed in the radial fibers of the transitional form of radial glia into mature astrocytes as well as partly in the corticofugal axonal bundles. EAAT3 immunoreactivity was robust in the apical dendrites of the pyramidal neurons in the marginal zone and cortical plate during corticogenesis, and decreased postnatally. In the individuals with type II lissencephaly, glutamate transporters were expressed in the extrusion of neuroglial tissue. Bundles of EAAT2-immunoreactive radial fibers were prominent in the specimens at 20 GW. Thus, glutamate transporters are differentially regulated during normal and impaired corticogenesis. Altered glutamate transporter expression in type II lissencephaly suggests that glutamate metabolism is involved in the formation of the normal cortex and contributes to the disorganized cortex seen in migration disorders.

Introduction

The glutamate transporter regulates the concentration of glutamate, the major excitatory neurotransmitter, at the synaptic cleft to prevent excessive stimulation of glutamate receptors in the mature brain [2], [24]. Glutamate receptors also localize to the central nervous system (CNS) before synaptogenesis; therefore, they may function in such developmental events as neurogenesis, cell migration, proliferation, differentiation, and axonal outgrowth [19], [32]. To date, five subtypes of sodium-dependent high-affinity glutamate transporters have been identified in human and animal tissues (animal isoforms are designated in parenthesis): EAAT1 (GLAST) [42], EAAT2 (GLT-1) [36], EAAT3 (EAAC1) [23], EAAT4 [11], and EAAT5 [1]. Although EAAT1 and EAAT2 are mainly localized to astrocytes in the postnatal brain, in rodents EAAT1 is expressed in proliferative zones and radial glia in the cerebral cortex [20], and EAAT2 is transiently expressed in developing axons [16]. Expression of EAAT3, which is primarily localized to neurons, is higher in the neonatal brain than in the adult rat brain [16]. Thus, glutamate transporters may have subtype-specific roles during CNS development, similar to glutamate receptors [19], [32].

Type II lissencephaly is classified as a migration disorder and is neuropathologically characterized by agyria–polymicrogyria patterns in the cerebral cortex [7]. It is found in the framework of several congenital muscle dystrophies including Walker Warburg's syndrome (WWS) [9], Fukuyama-type congenital muscular dystrophy (FCMD) [14], and muscle–eye–brain disease (MEB) [39]. Although these syndromes occur in different genetic backgrounds [4], [25], [50], recent studies have revealed that aberrant protein glycosylation of α-dystroglycan may be critical for formation of this type of cortical dysplasia [10]. Histopathological features of type II lissencephaly consist of focal absence of a glia limitans with derangement of radial fibers and extrusion of neuroglial tissue into leptomeninges [46]. Although glutamate receptors participate in the migration of neurons [26] and glutamate receptors and transporters demonstrate subtype-specific chronological changes during normal corticogenesis [3], [15], [16], studies regarding the pathophysiology of glutamate transporters have primarily addressed epileptogenisity [22] and have not been directed toward the migration disorders, including type II lissencephaly.

In the present study, we have examined the temporal and spatial expression of the glutamate transporter proteins EAAT1, EAAT2, and EAAT3 in the human frontal cortex of fetal (13 to 33 gestational weeks [GW]) and postnatal brains without neurological disorders and, in four cases, with type II lissencephaly (20 to 39 GW). Immunohistochemistry of reelin and vimentin was performed to discern Cajal–Retzius cells in the marginal zone or molecular layer, and radial glia, respectively [34], [47]. We found that expression of glial glutamate transporters changed with astrocytic maturation in the normal and disarranged neocortex. EAAT3 was preferentially expressed in the apical dendrites of the developing neurons. Subtype-specific chronological expression of the glutamate transporters during human corticogenesis and migration disorders are discussed.