Epilepsia, 50(12):2619–2628, 2009 doi: 10.1111/j.1528-1167.2009.02194.x
FULL-LENGTH ORIGINAL RESEARCH
Doublecortin expression in focal cortical dysplasia in epilepsy *Nisaharan Srikandarajah, *Lillian Martinian, *Sanjay M. Sisodiya, yWaney Squier, zIngmar Blumcke, xEleonora Aronica, and *Maria Thom *Department of Clinical and Experimental Epilepsy, Institute of Neurology, Queen Square, London, United Kingdom; yDepartment of Neuropathology, John Radcliffe Hospital, Oxford, United Kingdom; zDepartment of Neuropathology, University of Erlangen School of Medicine, Erlangen, Germany; and xDepartment of Neuropathology, Academic Medical Center, Amsterdam, and Epilepsy Institute of The Netherlands (SEIN), Heemstede, The Netherlands
SUMMARY Purpose: Doublecortin (DCX) is a microtubuleassociated protein with regulatory roles in radial and tangential migration of neurons during cortical development. In normal adult cortex there is restricted expression, and DCX is widely used as a marker of neurogenesis. Imperfect corticogenesis is thought to underpin many focal cortical pathologies in epilepsy surgical series, including focal cortical dysplasia (FCD). The aim was to study DCX expression patterns in such lesions compared to normal developing and mature cortex. Method: Cases of FCD types Ia (13) and IIb (4), pediatric hippocampal sclerosis (HS) (5), temporal lobe sclerosis (5), glioneuronal tumors (5), gray matter heterotopia (3), and control tissues (16) from a wide age range [20 gestational weeks (GW) to 85 years] were studied using immunohistochemistry to DCX.
Doublecortin (DCX) is a microtubule-associated phosphoprotein expressed by horizontal and radially migrating and differentiating neurons in the developing cortex (Gleeson et al., 1999). In adults, DCX has been employed as a reliable marker for new neurons in regions such as the dentate gyrus, where ongoing neurogenesis is recognized (Arvidsson et al., 2002; Brown et al., 2003; CouillardDespres et al., 2005). DCX-positive (DCX+) cells also Accepted May 8, 2009; Early View publication July 2, 2009. Address correspondence to Maria Thom, Division of Neuropathology, Department of Clinical and Experimental Epilepsy, Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom. E-mail:
[email protected] Wiley Periodicals, Inc. ª 2009 International League Against Epilepsy
Results: In controls and all epilepsy cases, perinuclear labeling of small round cells (SRCs) and satellite perineuronal cells was observed in both postmortem and surgical tissues. In FCD Ia up to the age of 4 years, prominent DCX-positive (DCX+), immature cells were present along the junction of layers I and II, with processes extending into the molecular layer. These cell types were not a significant feature in other pathologies, which showed multipolar DCX+ cells or labeling of dysmorphic cells throughout the cortex. Discussion: Persistent cellular DCX expression is confirmed in normal adult cortex. Characteristic expression patterns in layer II of FCD Ia could indicate delayed or abnormal cortical maturation rather than ongoing cytogenesis. This could be indicative of enhanced local cortical plasticity as well as a potential diagnostic feature of this type of pathology. KEY WORDS: FCD, Doublecortin, Layer II.
persist in the adult neocortex (Qin et al., 2000; Verwer et al., 2007; Xiong et al., 2008), although the cell lineage and physiologic functions of these populations are unclear. Experimental evidence suggests some may represent a reservoir of precursor cells (Tamura et al., 2007; Walker et al., 2007). Experimental injury models, including seizures, have also demonstrated recruitment of DCX+ proliferating cells toward the site of injury (Arvidsson et al., 2002; Hua et al., 2008; Jin et al., 2003). Mutations in the DCX gene give rise to laminar heterotopias or lissencephaly, which are associated with epilepsy (Gleeson et al., 1998). In addition, many focal neocortical lesions in surgical epilepsy series, including focal cortical dysplasias (FCD) types I and II and dysembryoplastic neuroepithelial tumor (DNT), are presumed to have a
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2620 N. Srikandarajah et al. maldevelopmental basis, and abnormal DCX expression has been demonstrated in some of these pathologies (Daou et al., 2005; Mizuguchi et al., 2002; Qin et al., 2000). Abnormal DCX expression could be a reflection of abnormal corticogenesis, delayed maturation, or, alternatively, enhanced plasticity or remodeling of residual neocortical DCX+-cell populations as a result of injury or seizures. We aimed to study the cytology and distribution of DCX+-cell populations in a spectrum of malformations in epilepsy, in particular FCD, to assess its potential contribution to pathogenesis as well as any diagnostic value in discriminating these types of pathologies in surgical practice.
Methods Case selection Epilepsy and control cases were selected from the neuropathology archives at the Institute of Neurology and the National Hospital for Neurology and Neurosurgery, London (United Kingdom); John Radcliffe Hospital, Oxford (United Kingdom); Academic Medical Center (University of Amsterdam; UVA), Amsterdam; University Medical Center in Utrecht UMCU (The Netherlands); and the University of Erlangen-Nuremberg (Germany). The study was approved by the respective ethics commit-
tees of these centers, where informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. For all surgical cases, patients underwent therapeutic surgical resection for refractory epilepsy and the pathologic tissue was surplus to diagnostic requirement. We studied 35 epilepsy cases in total as detailed in Table 1. Thirteen cases of FCD Ia with pathology features as previously described (Hildebrandt et al., 2005) were studied, four cases of FCD IIb, five pediatric cases of hippocampal sclerosis (HS) secondary to perinatal infarct, five adult cases of temporal lobe sclerosis adjacent to HS with architectural abnormalities in cortical layers II and III as previously described (Garbelli et al., 2006; Thom et al., 2000), five glioneuronal tumors (two complex and two nonspecific DNT and one ganglioglioma), and three cases of gray matter heterotopia [laminar/subcortical heterotopias (1) and nodular/periventricular heterotopia (2)]. Normal control tissues for comparison of DCX-staining patterns included fetal, neonatal, pediatric, and adult tissue as also detailed in Table 1. Immunohistochemistry Sections of 7-lm thickness from paraffin wax– embedded blocks were dewaxed in xylene and
Table 1. Details of cases and controls Number of cases Pathology group FCD Ia FCD IIb Pediatric temporal lobe epilepsy (HS with infarct)a Temporal lobe sclerosis (with HS) Glioneuronal tumors Heterotopia
13
Localization
Mean age (range)
4 5
Temporal (6), Frontal (3), Occipital (2), not specified (2) Frontal Temporal
10.2 years (2–32 years) 23.4 years (2–52 years) 10.6 years (0.3–17 years)
5
Temporal
35.2 years (27–47 years)
5 3
Temporal (3), Frontal (2) Subcortical (1), periventricular (2)
33.8 years (22–43 years) 37–83 years
Tissue type (surgical or PM) Surgical
PM
Ages Control groups Fetal
4
All regions
Pediatric
6
All regions including temporal lobe
Adult
3 3
Temporal Temporal
20 GW 23 GW 31 GW 36 GW 1 day 13 days 10 months 15 months 2 years 5 years 33–41 years 49–85 years
FCD, focal cortical dysplasia; GW, gestational weeks; HS, hippocampal sclerosis; PM, postmortem. a These cases were associated with a neonatal infarct. Epilepsia, 50(12):2619–2628, 2009 doi: 10.1111/j.1528-1167.2009.02194.x
All PM
PM
Surgical Surgical PM
2621 Layer II Doublecortin Expression in FCD rehydrated through graded alcohols and taken to water. Endogenous peroxidase was blocked in 3% hydrogen peroxide in water for 20 min. Sections were then microwaved at high power with antigen retrieval solution (Vector Labs, Burlington, CA, U.S.A.) for 12 min and then cooled for 20 min. After washing in water, sections were rinsed with phosphate-buffered saline (PBS) buffer (0.5% Tween 20 added) and incubated with polyclonal DCX (Abcam 1:4,000; 332 Cambridge Science Park, Cambridge, United Kingdom) overnight at 4°C. Labeling was detected using DAKO Envision horseradish peroxidase (Dako, Glostrup, Denmark). Staining was visualized using Dako DAB+ as a substrate. The staining was enhanced in copper sulfate briefly, and after washing sections were counterstained with hematoxylin dehydrated in graded alcohol, cleared in xylene, and coverslipped. The negative controls were treated identically except that primary antibody was omitted. Between all steps, sections were washed with PBS and 0.05% Tween 20. DCX Antibody was diluted in Dako ChemMate Diluent. Immunofluorescence Sections cut at 7 lm were dewaxed, rehydrated, and washed in water. Endogenous peroxidase was quenched using deionized water and 3% hydrogen peroxide. Sections were microwaved in antigen retrieval buffer from Vector Labs. After cooling, sections were incubated with primary antibody DCX from Abcam, and diluted 1:4,000 overnight at 4°C. Sections were incubated with secondary Dako Envision kit followed by Cy3 Tyramide Signal Amplification (PerkinElmer Life and Analytical Sciences, Boston, MA, U.S.A.). After washing, the sections were quenched with 1% hydrogen peroxide for 15 min to prevent any deposited tyramide combining with the second tyramide signal that followed. After washing, protein blocking was carried out with normal horse serum using ImmPRESS kit (Vector Labs) followed by incubation with primary monoclonal antibodies GFAP (1:40; Dako), NeuN (1:1,000; Chemicon, Temecula, CA, U.S.A.), and Calbindin D-28K (1:2,000; Sigma, St. Louis, MO, U.S.A.) for 1 h at room temperature. Sections were incubated with anti-mouse ImmPRESS kit followed by TSA (Perkin Elmer Life and Analytical Sciences). Goat polyclonal DCX (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.) was used in combination with rabbit polyclonal GFAP-delta (1:5,000; Chemicon International); the detection system for DCX was Avidin Biotin kit from Santa Cruz followed by fluorescein TSA, and the detection system for GFAP-delta was anti Rabbit ImmPRESS kit from Vector followed by Cy3 TSA (Perkin Elmer). Sections were mounted on Vectashield with Dapi (Vector Laboratories) and visualized with a Zeiss LSM 510 meta confocal laser microscope (Carl Zeiss AG, Oberkochen, Germany).
Results Controls Fetal cases At 20 gestational weeks (GW), islands of immature cells in the germinal matrix showed positive cytoplasmic labeling for DCX. In the developing cortex at this stage doublecortin-positive (DCX+) cells were frequent in the external granule cell layer, less frequent in the marginal zone, and more numerous through the full thickness of the cortical plate (Fig. 1A). At 23 GW, DCX+ cells showed a more distinct bipolar or fusiform morphology migrating through the subplate. An additional population of DCX+ small round cells (SRCs) was also noted in the subplate and cortical plate, in addition to bipolar cells, with no visible cell processes. In the cortical plate, elongated, fusiform DCX+ cells with a mainly radial orientation were frequent, interspersed between DCX-negative mature neurons. By 36 GW, DCX+ cells were still easily identified in the white matter and cortex but diminished in number compared to 23 weeks; these remaining DCX+ fusiform cells showed a uniform distribution from layers II to VI (Fig. 1B). Neonatal cases At 1 day the features were similar to 36 GW. Bipolar, fusiform, DCX+ cells persisted within the white matter (Fig. 1F), in the vicinity of the residual periventricular germinal matrix and throughout cortical layers II–VI of the temporal lobe neocortex (Fig. 1C). At this age these cells were more numerous in the outer cortical layers (I–IV) than in deeper cortex. In general, their processes were aligned in a radial axis, although occasional cells with a more horizontal orientation were observed. Double-labeling with NeuN at this stage confirmed that a proportion of DCX+ cells were also NeuN-positive (Fig. 1G). The immunostaining pattern, cytomorphology, and distribution at 13 days were similar to those at 1 day, although a further reduction in DCX+ cell number was apparent (Fig. 1D,E). By 10 months there was a marked diminution in the number of DCX+ cells in the neocortex. In the parahippocampal gyrus, residual DCX+ cells with bi- or unipolar processes remained present in layer II. By 15 months a near absence of fusiform, bipolar cells in the neocortex was noted as well as in the white matter. In the parahippocampal gyrus in layer II, occasional residual DCX+ small cells with processes persisted. By age 2 and 5 years, DCX+ cells with cellular processes were seen only infrequently. DCX+ SRC, however, were present in the white matter at all stages, and to a lesser extent in the cortex. Adult controls In surgical and postmortem cases, DCX+ cells with processes or bipolar morphology were virtually absent. Epilepsia, 50(12):2619–2628, 2009 doi: 10.1111/j.1528-1167.2009.02194.x
2622 N. Srikandarajah et al.
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Figure 1. Doublecortin (DCX) in controls. (A) Twenty gestational weeks: doublecortin-positive (DCX+) cells are noted in the external granule cell layer, with fewer cells in the marginal zone and more frequent cells in the upper part of the forming cortical plate. (B) Thirty-six gestational weeks: bipolar and fusiform DCX+ cells are clearly seen in cortical layers I and III with the morphology of migrating cells. (C) One day neonatal: bipolar DCX+ cells persist in the maturing cortex, mainly with a radial alignment as shown in cortical layers II and III. (D) Thirteen days neonatal: there is an overall impression of reduced density of DCX+ cells compared to developmental stages in the upper cortical layers. (E) Thirteen days neonatal: DCX+ cells in layer II are interspersed between mature neurons in layer II and retain a bipolar morphology. (F) One day neonatal: bipolar DCX+ cells are present in the subcortical white matter. (G) One day neonatal: DCX+ positive cells in the white matter (red) show occasional colocalization with neuronal nuclear antigen NeuN (green) as evidence of neuronal differentiation. (H) Adult control: small round cells (SRCs) show cytoplasmic labeling with DCX with no cytoplasmic processes; a perineuronal satellite location of a DCX-positive cell is shown (top left) in the neocortex. Bar in D (for A–D) 100 lm, in E (for E–G) 50 lm, and in H 35 lm. Epilepsia ILAE There was labeling of SRC in cortical layer I, white matter, cortex, and hippocampus. A proportion of the cortical SRC was in a satellite location in proximity to unstained cortical neurones (Fig. 1H). Weak cytoplasmic labeling of larger neurons was observed occasionally, but processes were rarely identified and DCX+ cells with a multipolar or astrocytic morphology were not seen; double-labeling for DCX and GFAP showed no colocalization. Epilepsy cases FCD Ia The histologic features were characterized by abnormal cortical laminar architecture, with an exaggerated radial alignment and persistent microcolumnar alignment of neurons in midcortical layers but an absence of balloon cells or dysmorphic neurons as seen in FCD II (Hildebrandt et al., 2005). In some cases, hypercellularity of layer II with an increased number of small, immature cells with small hyperchromatic nuclei was noted (Fig. 2A). In a single case, additional depletion of neurons in layer II was confirmed on NeuN, with clustering of remaining neurons (Fig. 2H). In all cases DCX immunohistochemistry showed labeling of SRC Epilepsia, 50(12):2619–2628, 2009 doi: 10.1111/j.1528-1167.2009.02194.x
in the cortex and white matter similar to that in pediatric and adult controls, with occasional weak cytoplasmic staining of the cell bodies of small and large pyramidal neurons. A striking additional finding in five cases from patients of 2 years was a distinct band of intensely labeled DCX+ cells at the border between cortical layers I and II (Fig. 2B). These were mainly small cells, many showing one or more DCX+ cytoplasmic processes extending into the molecular layer (layer I) with a radial orientation (Fig. 2C); focal aggregation of these processes was also observed (Fig. 2D). Small, multipolar DCX+ cells (Fig. 2E) as well as occasional cells with a more horizontal alignment, and occasional bipolar cells were also present. In the single case with evidence of neuronal depletion from layer II (Fig. 2H), aggregation and more prominent horizontal alignment of DCX+ cells in the remnants of this cell layer were appreciated (Fig. 2I, 2J). DCX+ layer II cells were noted in FCD Ia cases involving the frontal and temporal lobes but less prominent in one case from the occipital lobe. DCX+ layer II cells were less frequent in FCD Ia cases from patients ages 3 and 4 years, and not identified in older ages. Double-labeling with NeuN
2623 Layer II Doublecortin Expression in FCD A
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Figure 2. Focal cortical dysplasia type Ia (FCD Ia) and temporal lobe sclerosis. (A) FCD Ia, age 2: Hematoxylin and eosin (H&E) stained section of layers I and II showing frequent scattered small cells with hyperchromatic nuclei but little cytoplasm scattered through layer II. (B) FCD Ia, age 2: doublecortin (DCX) immunostaining showing frequent small DCX positive (DCX+) cells at the interface between layers I and II. Many cells show extension of cellular processes into layer I. (C) FCD Ia, age 2: a further case highlighting layer-II DCX+ cells with cellular processes extending radially into layer I. (D) FCD Ia, age 2: layer-II DCX+ cells with clustering of processes was a further finding. (E) FCD Ia, age 2: layer II DCX+ cells in some cases appeared more disorganized and randomly oriented. (F) FCD Ia, age 2: double labeling in layer II with DCX (red) and NeuN (green) did not show any colocalization. (G) FCD Ia, age 2: no colocalization between DCX (red) and GFAP (green) was observed in layer I/II interface. (H) FCD Ia with evidence of patchy neuronal loss from layer II compared to the preserved pyramidal cells in deeper cortical layer III as confirmed with NeuN immunostaining. (I) FCD Ia (case as in H) with a prominent horizontal orientation of residual DCX+ cells at layer I/II interface. (J) FCD Ia (case as in H): prominent grouping or aggregation of residual DCX+ cells in layer II was also noted. (K) Temporal lobe sclerosis: NeuN reveals loss of cells from layer III with clustering of small cells in the superficial part of layer II imparting an abnormal architecture. Bar shown in A is equivalent to 100 lm in (A, H), 35 lm in (C, F, G, I, J), 50 lm in (D, E), and 200 lm in (B, K). Epilepsia ILAE
(Fig. 2F), GFAP (Fig. 2G), and calbindin showed no colocalization with DCX+ layer II cells. DCX+ cells of similar morphology in layer II were seen only rarely in controls and other epilepsy pathologies (Fig. 3). FCD IIb The histopathologic features of these cases fulfilled the criteria for FCD IIb (Palmini et al., 2004). A proportion of dysmorphic and balloon cells showed strong cytoplasmic labeling with DCX through all cortical layers, but with a dominance in the lower cortex (Fig. 4A). Scattered hypertrophic DCX+ multipolar cells and multinucleate balloon cells were a prominent finding
(Fig. 4B). In addition to intensely labeled DCX+ SRCs and perineuronal satellite cells (Fig. 4C), a further observation was ‘‘cell-embracing’’ of weakly labeled neurons by the processes of large, multipolar, intensely labeled DCX+ cells in proximity (Fig. 4D,E). Doublelabeling confirmed that a proportion of DCX+ balloon cells were GFAP (Fig. 4F) and GFAP-delta positive, but no colocalization with NeuN (Fig. 4I) or calbindin was seen. In pediatric FCD IIb (ages 2 and 5 years) DCX+ layer II cells, as described in FCD I cases, were noted only occasionally, either within the region of dysplasia or in the adjacent cortex with normal architecture (Fig. 3C). Epilepsia, 50(12):2619–2628, 2009 doi: 10.1111/j.1528-1167.2009.02194.x
2624 N. Srikandarajah et al.
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Figure 3. Expression of doublecortin (DCX) in layer II in epilepsy cases and controls. (A) Focal cortical dysplasia type Ia (FCD Ia) 2 years: tufted and semilunar DCX-positive (DCX+) cells with processes extending into layer I. (B) Thirty-one weeks gestation control; Temporal cortex: bipolar fusiform DCX+ cells are seen but no enhanced labeling of cells at the interface of layers I and II is seen or cells of a similar morphology to that of FCD Ia cases. (C) FCD IIb, age 2 years: the adjacent frontal cortex to the dysplasia shows scattered cells in layer II with radial processes, of similar morphology to those in FCD Ia cases. (D) Ten month control, fusiform gyrus (no epilepsy): there are scatted cells with weakly labeled processes extending into the molecular layer. (E) Temporal lobe epilepsy with hippocampal sclerosis (HS) 1 year: there are no distinctive DCX+ cells with processes in layer II. (F) Surgical control 2 years (no epilepsy): no distinctive DCX+ cells with processes in layer II are evident. Bars in A, C, E, F = 100 microns, in B, D = 10 microns. Epilepsia ILAE
Pediatric HS These cases were characterized by varying degrees of HS and an adjacent neonatal cystic–gliotic cortical infarct. In the region of the hippocampus, variable DCX+ labeling of granule cell neurons was noted. In the white matter of the hippocampus and parahippocampal gyrus, labeling of SRC was noted in addition to cells with multipolar glial-like morphology in the superficial cortex (away from the area of infarction) as well as in the hilar region of the hippocampus (Fig. 4G). DCX+ perineuronal satellite cells were noted, particularly at the cortical white matter Epilepsia, 50(12):2619–2628, 2009 doi: 10.1111/j.1528-1167.2009.02194.x
junction. DCX+ layer II cells in the parahippocampal gyrus or neocortex were not identified (Fig. 3E). Adult temporal lobe neocortical sclerosis These cases were all characterized by neuronal loss and gliosis in layers II and III, with clustering of remaining neurons in outer layer II as described previously (Garbelli et al., 2006; Thom et al., 2000) (Fig. 2K). Labeling of small, multipolar cells in layers I and II (Fig. 4H) with DCX was observed, some in proximity to neurons, with cytoplasmic extensions enveloping the cell body. These
2625 Layer II Doublecortin Expression in FCD
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Figure 4. Focal cortical dysplasia type IIb (FCD IIb), pediatric hippocampal sclerosis (HS), and temporal lobe sclerosis. (A) FCD IIb: frequent cytoplasmic labeling of variable intensity of dysmorphic cells with doublecortin (DCX) is seen throughout cortex in region of dysplasia. (B) FCD IIb: intense cytoplasmic labeling of multipolar, hypertrophic balloon cell in FCD IIb by DCX was a frequent finding. (C) FCD IIb: prominent labeling of perineuronal satellite cells and small round cells through the region of dysplasia as well as adjacent cortex was noted with DCX. (D) FCD IIb: enlarged satellite cell with processes enveloping adjacent neuron was observed in FCD IIb cases. (E) FCD IIb: as for D. (F) FCD IIb: colocalization of DCX (red) and GFAP (green) was demonstrated in a proportion of balloon cells. (G) Pediatric HS: multipolar DCX positive (DCX+) cell in the white matter. (H) Temporal lobe sclerosis: frequent DCX+ multipolar cells were present in layer II. (I) FCD IIb: colocalization of DCX (red), as demonstrated here in a positive balloon cell, and NeuN (green) was not observed. Bar as shown in A equivalent to 100 lm (A), 75 lm in (C), 50 lm in (B), 35 lm (D, E, G–I), and 20 lm in (F). Epilepsia ILAE
multipolar cells appeared relatively restricted to the abnormal upper cortex and were not noted in the deeper cortex or white matter, which showed extensive fibrillary gliosis on GFAP. Colocalization between GFAP-delta and DCX was not observed. Layer II DCX+ cells, as seen in FCD Ia, were not a feature.
the astroglial or oligodendroglial tumoral component. Clusters of DCX+ multipolar cells in the adjacent upper cortex were noted, as seen in temporal lobe sclerosis cases. Double-labeling with DCX and CD34 did not show any colocalization of expression. Layer II DCX+ cells, as seen in FCD Ia, were not a feature.
Glioneuronal tumors In the adjacent cortex, intensely labelled DCX+ SRC and perineuronal satellite cells were observed. Within the lesions, labeling of occasional, larger, atypical neurons as well as eosinophilic granular bodies was noted, but not of
Heterotopia In all three heterotopia cases, similar DCX-staining patterns were noted in the heterotopia and overlying cortex, as observed in the adult controls. Cytoplasmic labeling of SRC and perineuronal cells with infrequent Epilepsia, 50(12):2619–2628, 2009 doi: 10.1111/j.1528-1167.2009.02194.x
2626 N. Srikandarajah et al. labeling of neurons and processes was observed. No labeling of cells with astroglial morphology was identified. Layer II DCX+ cells, as seen in FCD Ia, were not a feature.
Discussion Although largely recognized for its role in neuronal migration during cortical development (Francis et al., 1999) persistent, albeit reduced, expression of DCX, is documented in the maturing and adult cortex (Nacher et al., 2001; Verwer et al., 2007; Walker et al., 2007). Its physiologic function postdevelopment as well as its cellular localization in human tissues, is less explored but speculated to include roles in the ongoing neuronal and dendritic plasticity (Meyer et al., 2002; Nacher et al., 2001). In the dentate gyrus, DCX has been used as a reliable marker for adult neurogenesis (Brown et al., 2003; Couillard-Despres et al., 2005; von Bohlen Und Halbach, 2007), although concern regarding reexpression in mature neurons under certain circumstances has been noted (Gould, 2007). Experimental studies have demonstrated increased DCX+ neurons in the region of an infarct, and it has been proposed that these cells have migrated to this region (Jin et al., 2003) or could be generated locally from precursor cells (Hua et al., 2008). Under pathologic conditions such as epilepsy, it is plausible that any altered DCX expression, either arising as a result of abnormal cortical development or an effect of the disease process or seizures, could be indicative of aberrant neurogenesis, cortical reorganisation, altered connectivity, and neuronal–glial interactions, which could be of functional significance. In this study we have demonstrated distinct DCX-expression patterns in cortical malformative lesions associated with epilepsy in comparison to normal cortex. Our studies of DCX expression at varying stages of normal cortical development are largely in agreement with previously studies in both humans and animals (Francis et al., 1999; Meyer et al., 2002). However, a novel observation was ongoing DCX expression in human neonatal cortex. In the first few weeks of life, bipolar fusiform cells with the morphology of immature and migrating neurons were identified within the cortex, similar to those seen during development. Focal NeuN coexpression was shown in support of neuronal differentiation, and a rapid decline in number was observed over the first months of life with cortical maturation. Within the fetal and neonatal controls we also observed a gradient of increased numbers of these cells in outer cortical layers as reported previously, speculated to reflect altered microtubule dynamics following transition from migratory to stationary mode (Gleeson et al., 1999; Meyer et al., 2002). A striking further finding in our series was the identification of extensive layer II DCX+ cells. These were most evident in patients with epilepsy and FCD Ia malformations of 2 years of age, particularly in a frontal–temporal Epilepsia, 50(12):2619–2628, 2009 doi: 10.1111/j.1528-1167.2009.02194.x
location. Double-labeling demonstrated a lack of colocalization with NeuN, GFAP, and calbindin, supporting an undifferentiated or immature phenotype. In other pathologies studied, including pediatric FCD IIb and HS, in addition to nonepilepsy controls spanning a wide age range from development through maturation, we did not observe similar DCX expression patterns at any time point, with only isolated cells of similar morphology identified. Previous studies that have included similar aged controls have also failed to identify these cell types (Mizuguchi et al., 2002; Qin et al., 2000; Verwer et al., 2007). They are reminiscent of the small immature DCX+ cells described in layers II and III of the piriform cortex and entorhinal cortex of the adult rat that also coexpresses PSA-NCAM (Nacher et al., 2001). These have been described variably as neurogliaform, ‘‘tangled’’ (Gomez-Climent et al., 2008), and semilunar cell types, the latter having a globular cell body and two or three apical dendrites extending into layer I and a basal axon (Nacher et al., 2001). Similar layer-II DCX+ cells, aligned at the junction of layers I and II, have also been reported in primates and larger mammals, including the adult guinea pig, described throughout the allocortex and isocortex including temporal and parietal lobes and often occurring in clusters (Xiong et al., 2008). The precise nature of these immature cells in mature mammalian cortex is unknown. Studies suggest that they are in a prolonged state of immaturity, functionally dormant, lacking synaptic contacts and that they do not show expression of markers of glial cells or mature neurons, including NeuN, or MAP2 (Gomez-Climent et al., 2008), although some evidence for c-aminobutyric acid (GABA)ergic neuronal differentiation has been demonstrated (Xiong et al., 2008). Studies confirm a dramatic decline in their number in animals with aging (GomezCliment et al., 2008; Xiong et al., 2008), which suggests that they undergo cell death or possibly differentiation. It is considered that layer-II DCX+ cells in animals are unlikely to represent newly acquired or migrated cells during adulthood but that they are generated prenatally (GomezCliment et al., 2008) and represent a reservoir of cells contributing to ongoing cortical plasticity (Bonfanti, 2006; Xiong et al., 2008). It has been suggested that they could be vulnerable under pathophysiologic conditions. In our series layer-II DCX+ cells were identified mainly in pediatric FCD Ia cases and were not present in significant numbers in other epilepsy pathologies or normal cortex from similar ages. One consideration is that varying tissue preservation, fixation, and postmortem decay could affect DCX immunostaining as has been suggested in previous work (Boekhoorn et al., 2006). However, because distinct labeling of bipolar migrating neurons for DCX was clearly demonstrated in postmortem controls (Fig. 1), this seems unlikely. Furthermore, in FCD Ia cases, layer-II hypercellularity, including small dark immature-appearing cells, was noted on hematoxylin and eosin (H&E)
2627 Layer II Doublecortin Expression in FCD staining, likely corresponding to DCX+ cells. DCX could, therefore, represent a specific layer marker for the confirmation of early FCD Ia cases, a diagnosis that is acknowledged to be more difficult to compare objectively to more overt dysplasias, such as FCD IIb (Palmini et al., 2004). An age-related decline of layer-II DCX+ cells in FCD Ia between 2 and 4 years was appreciated in our series. Layer II is the last lamina to be formed, and it is plausible that delayed cortical maturation results in recruitment or failure of elimination of DCX+ cells, which thereafter differentiate or die. Their prolonged presence may highlight a window of early enhanced local cortical plasticity, which could be relevant to the development of abnormal local layer II networks, and promote seizures. The exact nature and fate of layer-II DCX+ cells in epilepsy remains speculative including any capacity for neuronal or glial differentiation. In our adult epilepsy group with temporal lobe sclerosis, characterized by layer-II architectural abnormalities, including clustering of mature NeuN-positive neurons (Garbelli et al., 2006; Thom et al., 2000), DCX+ multipolar cells were seen relatively restricted to this region. We did not observe generalized DCX labeling of astrocytic gliosis, for example, in the temporal lobe white matter, which is supported by previous experimental studies of DCX expression in gliosis (Couillard-Despres et al., 2005). Furthermore, no overlap of DCX and delta-GFAP expression was seen, an isoform of GFAP expressed in multipotent progenitor cell glial types (Roelofs et al., 2005) and upregulated in epilepsy malformative pathologies (Martinian et al., 2008); or CD34, a stem-cell marker expressed in glioneuronal tumours (Blumcke et al., 1999). These findings highlight the possibility of several immature cell types contributing to epilepsy lesional pathologies, of which DCX+ cells are one, the nature and lineage of which requires further investigation. Previous studies of DCX in the adult human cortex have also reported persistent expression in small cells, similar to the ‘‘SRC’’ and satellite cells we describe in epilepsy cases as well as controls; such cells have been previously interpreted as ‘‘glia’’ but without further qualification (Mizuguchi et al., 2002; Qin et al., 2000). In the adult cortex of rat, similar DCX+ cells in a perineuronal territory were reported throughout the cortex, with wrapping of cell processes around adjacent NeuN-positive neurons (Tamura et al., 2007). These satellite cells in animals coexpress NG-2 and incorporate BrdU, and are proposed to represent cortical multipotential precursors, giving rise to cells of neuronal and glial lineages (Tamura et al., 2007; Walker et al., 2007). Furthermore, in a recent study of DCX expression in Alzheimer autopsy and surgical epilepsy tissue, a proportion of glia, predominantly in layer I and cortical-white matter junction, were colabeled
with GFAP with processes enveloping neurons (Verwer et al., 2007). One possibility, in line with these previous studies, is that residual DCX+ SRC in adult cortex represent a persisting reservoir of progenitor cells with capacity for glial and neuronal differentiation, related to the NG-2 cells or polydendrocytes (Nishiyama et al., 1999). In dysplasia cases, DCX+ balloon cells in FCD IIb colocalized with GFAP, as evidence of glial differentiation. The proximity of SRC with neurons and the demonstration of DCX+ cell processes enveloping neurons in epilepsy pathologies, also draw comparisons with descriptions of NG2/DCX+ cells described in animal models (Tamura et al., 2007). An increase in perineuronal satellite cells and perivascular SRCs in the white matter in conventional stained sections is a frequent observation in surgical resections in epilepsy and previously encompassed within the spectrum of changes of ‘‘microdysgenesis’’ or mild dysplasia (Armstrong, 1993; Kasper et al., 1999). NG2 cells are known to receive synaptic inputs from neurons (Wigley et al., 2007), are closely associated with interneurons (Mangin et al., 2008) with potential roles in brain repair (Komitova et al., 2006; Nishiyama, 2007), and their numbers have been shown to increase following experimental seizures (Wennstrom et al., 2003). These DCX+ cell types, therefore, warrant further study in focal epilepsies. In summary, further investigation into the nature of residual DCX+ cells and their enhanced expression patterns in focal epilepsy malformative pathologies is of interest for their potential in the establishment of pathological neuronal–glial interactions. This current study highlights that the identification of excessive layer-II DCX+ cells may be a useful diagnostic feature of pediatric FCD Ia.
Acknowledgments Lillian Martinian is supported by the MRC (Grant G79059). EA is supported by National Epilepsy Fund NEF 05-11 and NF 09-5. We confirm that we have read the journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Disclosure: None of the authors has any conflict of interest to disclose.
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