Immunohistochemical localization of the cell adhesion molecules Thy ...

Molecular Psychiatry (1999) 4, 46–52  1999 Stockton Press All rights reserved 1359–4184/99 $12.00

ORIGINAL RESEARCH ARTICLE

Immunohistochemical localization of the cell adhesion molecules Thy-1 and L1 in the human prefrontal cortex: patients with schizophrenia, bipolar disorder, and depression MJ Webster1, MP Vawter2 and WJ Freed2 1

Stanley Foundation Research Program, Neuroscience Center at St Elizabeths, Washington, DC 20032; 2NIDA IRP, 5500 Nathan Shock Dr, Baltimore, MD 21224, USA L1 and Thy-1 are members of the immunoglobulin (Ig) superfamily of cell adhesion molecules (CAMs) that are vital for normal neural development. Abnormalities in CAM expression could lead to the histological abnormalities that have previously been described in the frontal cortex of patients with schizophrenia. A postmortem immunohistochemical study of L1 and Thy-1 in the normal human prefrontal cortex revealed positive immunostaining of axons in all layers of the cortex. Quantifying the intensity of immunostaining in the prefrontal cortex of patients with schizophrenia, bipolar disorder and depression failed to reveal any significant differences when compared to that of normal controls. Keywords: cell adhesion molecule; immunohistochemistry; immunoblot; bipolar disorder; schizophrenia; depression; prefrontal cortex

Introduction Several cytoarchitectural abnormalities have been described in the frontal and in medial temporal cortical regions of individuals with schizophrenia. Disturbed laminar organization in the frontal and entorhinal cortices,1–3 misplacement of NADPH-diaphorase positive neurons in both frontal and temporal cortices,4,5 abnormally high neuron density in the frontal cortex6 and abnormal size and orientation of hippocampal neurons7,8 have all been suggested to support the developmental origin of abnormal brain function in schizophrenia. Cell adhesion molecules (CAMS) of the Ig superfamily play an important role in the development of the brain. An abnormality in expression of one of the CAMs could result in the histological abnormalities described in the brains of individuals with schizophrenia.9 The Ig superfamily includes CAMs ranging from those with only one Ig domain (eg P0, Thy-1), to those with as many as six Ig domains and two to five fibronectin-type III domains (eg N-CAM, L1). Among this family of molecules, both Thy-1 and L-1 expression in the CNS is almost exclusively axonal. Both Thy-1 and L1 are of potential interest in relationship to major mental illnesses. In the CSF of

Correspondence: Dr MJ Webster, Stanley Foundation Research Program, NIMH Neuroscience Center at St Elizabeths, 2700 Martin Luther King, Jr Blvd, SE Washington, DC 20032, USA. E-mail: websterm얀dirpc.nimh.nih.gov Received 27 January 1998; revised and accepted 1 June 1998

patients with schizophrenia and mood disorder there is an increase in N-CAM as compared to normal controls,10,11 and schizophrenic subjects show an increase in a 105–115 kDa N-CAM isoform in hippocampus and prefrontal cortex.12 Conversely, there is a decrease in the 140-kDa isoform of L1 in the CSF of patients with schizophrenia.10,13 Since L1 is not a significant constituent of serum proteins,10 it is presumed that the L1 decrease detected in the CSF must reflect concentrations or turnover somewhere in the brain. Interestingly, Thy-1 maps to the same region of chromosome 11 as both N-CAM and the D2 dopamine receptor,14–16 both of which may be increased in schizophrenia (for review see Seeman, 199117). Thus, both Thy-1 and L1 are of potential interest in relationship to schizophrenia. Therefore, we have examined the immunohistochemical localization of Thy-1 and L1 in the prefrontal cortex of patients with major mental illnesses and in normal controls. While several previous studies have described the distribution of Thy-1 in the normal adult human cortex,18–20 this is the first description of L1 in the adult human cortex.

Materials and methods Tissue Frozen postmortem samples of dorsolateral prefrontal cortex were obtained from the Stanley Foundation Neuropathology Consortium. The consortium collection consists of 15 subjects with schizophrenia, 15 with bipolar disorder, 15 with major depression without

Immunohistochemical localization of Thy-1 and L1 MJ Webster et al

psychotic features and 15 normal controls. All groups are matched for age, sex, race, and postmortem interval (Table 1). One hemisphere from each brain was rapidly frozen in isopentane and dry ice and stored at −70°C until further processing. Primary antibodies A mouse anti-Thy-1 monoclonal antibody (Clone F1542-1) was obtained from Serotec (Kidlington, UK). A mouse anti-L1 monoclonal antibody (neuro 4.1.1.3) was obtained from Becton Dickinson, NC, USA). Western blot analysis To determine the specificity of monoclonal Thy-1 and L1 antibodies, samples of occipital cortex were fractionated into cytosolic and particulate membrane fractions21 (with slight modifications12) and separated by standard SDS-PAGE22 on 7.5% gels under denaturing and reducing conditions (L1 and Thy-1) or non-denaturing conditions (Thy-1). The gels were blotted to nylon-supported nitrocellulose membranes (Amersham Corp, IL, USA) and monoclonal anti-Thy-1 or L1 was incubated overnight at 1:1000 at 4°C. Immunoabsorption experiments were conducted with a bacterial extract containing either a recombinant glutathione S-transferase (GST) protein as a control or the entire human L1 sequence23 inserted into a plasmid GST expression vector (pGEX; Pharmacia, Uppsala, Sweden) to further determine monoclonal L1 antibody specificity. Either recombinant GST protein or the entire human L1 protein fused to GST were used as immunoabsorbent.12 Cortical tissue samples were immunoblotted with the L1 monoclonal antisera following preabsorption with L1 fusion protein, and compared to L1 antisera following the control GST absorption. Immunohistochemistry Frozen blocks from the middle frontal gyrus were sectioned in the coronal plane at 14 ␮m thickness, mounted on subbed slides, and were stored at −70°C. On the day of the experiment the sections were thawed, air dried and fixed in 4% paraformaldehyde for 10 min, washed in PBS, preincubated in 10% normal horse serum and incubated at 4°C overnight with Table 1

Stanley Foundation Neuropathology Consortium 15 15 Schizophrenia Bipolar disorder

Age (yrs) Sex Race PMI (h)

15 Depression without psychosis

15 Normal controls

48.1 (29–68)

44.2 (25–62)

42.3 (25–61)

46.5 (30–65)

9 M, 6 F

9 M, 6 F

9 M, 6 F

9 M, 6 F

15 W

14 W, 1 B

27.5 (7–47)

23.7 (8–42)

13 W, 2 As 14 W, 1 B 33.7 (12–61)

32.5 (13–62)

the primary antibody (Thy-1 diluted 1:1000; L1 diluted 1:100). The sections were then rinsed in PBS and incubated for 1 h in biotinylated sheep anti-mouse IgG (preabsorbed against rat, rabbit and human IgGs; Amersham Corp; 1:80) at room temperature. They were then rinsed in PBS before the final 1-h incubation in the streptavidin-fluorescein conjugate (Amersham Corp; 1:80). Analysis Quantification of the immunostaining was performed in accordance with previously published criteria.24–28 Thus, each experiment was performed in a single session to ensure that all sections were exposed to the same conditions. Prior to each experiment antibody concentrations were titrated to ensure that the signal intensity was linearly related to the antigen concentrations. The dilution used in the experiment was chosen from the center of the linear range. Mean density of immunostaining was measured using the Macintosh-based analysis program (NIH IMAGE 1.33). The stained sections were studied with a 20× objective using a Zeiss Axiophotmicroscope, and a DAGE-MTI SIT68 camera. Fluorescence intensity was measured in relative gray scale units with the camera sensitivity adjusted so that the range of average mean fluorescence intensity for each antibody was approximately 120–250 gray scale units. For each subject, five traverses were made through the cortex and within each traverse five measurements were taken. Each measurement was from an area 400 ␮m2. The first measurement was from layers I/II, the second from layer III, the third from deep III and IV, the fourth from layers V/VI, and the fifth from the white matter. Each complete set of measurements was performed in a single session, to minimize sources of variability related to camera and microscope settings, and fading of the slides.25,27

Results The Western blot analysis confirmed the specificity of the antibodies employed in this study. Thy-1 immunoblots revealed a single protein band at 25 kDa (Figure 1). The Thy-1 antibody was sensitive to the denaturing status of samples analyzed by Western immunoblot (Figure 1). Non-denaturing conditions gave a strong signal (Figure 1a) while denaturing the antigen apparently destroyed the immunoreactivity (Figure 1b). The L1 immunoblots of soluble and membrane fractions revealed two major protein bands at 200 and 140 kDa (Figure 2). Immunoabsorption experiments with L1 fusion protein blocked binding of the monoclonal antibody to both the L1-200 and L1-140 proteins by greater than 95% compared to preabsorption with GST protein (not shown). Examination of Thy-1 immunostained sections of frontal cortex revealed very little Thy-1 staining in the white matter, but as axons enter the gray matter they became intensely Thy-1 positive (Figures 3,4). The fluorescent bands of fibers radiated into the gray mat-

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Figure 1 Western blot analysis of homogenates from human cortex analyzed using monclonal anti-Thy-1 (Serotec). The gel for immunoblot (a) was run under non-reducing and nondenaturing conditions, while the gel for immunoblot (b) was run under reducing and denaturing conditions. Lanes 1 and 2 are occipital cortex membrane preparations (10, 20 ␮g) and lanes 3 and 4 are cytosolic preparations (5, 30 ␮g).

Figure 2 Western blot analysis of homogenates from human cortex analyzed using monoclonal anti-L1 (Becton Dickinson). Lanes 1–4: A cytosolic brain preparation was titered against varying dilutions of anti-L1 with a surf-blotter apparatus (Idea Scientific, Minneapolis, MN, USA). Lanes 5, 6: A membrane cortex preparation was immunoblotted in lane 5 and a cytosolic preparation in lane 6. Dilutions of antiL1 are shown from 1:50–1:2000.

Figure 3 Photomicrograph illustrating Thy-1 positive immunofluorescent bundles of fibers entering the deep layers of the cortex (bottom) and forming a dense plexus of fibers in the superficial layers (top). Scale bar = 100 ␮m.


49

Figure 4 Photomicrographs illustrating Thy-1 (a,b) and L1 (c,d) immunofluorescence in the frontal cortex. (a) A dense plexus of Thy-1 immunoreactive fibers in superficial layers. (b) Immunofluorescent fiber bundles in deep layers. (c) Diffuse L1 immunoreactivity in cortical neuropil surrounding ‘ghosts’ of cell bodies (large arrows). (d) Fine L1 immunoreactive fibers in white matter (large arrows). The small arrows in each photograph indicate autofluorescent lipofuschin pigment granules. Scale bars = 50 ␮m.

ter, forming a dense plexus of individual fibers in the superficial layers (Figure 4a). The intensity of fluorescence was greatest in the deep cortical layers and gradually declined to lower levels in layers I/II (Figure 5). Although the control group showed slightly lower levels of staining intensity than the affected groups, there was no significant effect of diagnosis at any level

in the cortex (F (12 200) = 1.02; P = 0.43). Numerous autofluorescent lipofuchsin pigment granules were also noted in the cortex of all cases (arrows in Figures 3, 4). Immunostaining with the L1 antibody revealed a diffuse pattern of staining in the neuropil of the cortex with ghosts of unstained cells throughout all layers (Figure 4c). Very fine fibers were immunofluorescent


50

Figure 5 Intensity of Thy-1 immunoreactivity in human prefrontal cortex as a function of cortical layers. The intensity was not different between diagnostic groups (P = 0.43).

in the white matter (Figure 4d). The intensity of fluorescence was low in the white matter and somewhat greater in the gray matter but was evenly distributed across all cortical layers (Figure 6). There was no significant effect of diagnosis on the intensity of L1 immunofluorescence (F (12 200) = 0.54, P = 0.89).

Discussion The present study demonstrated that although the cell adhesion molecules Thy-1 and L1 are abundantly expressed throughout the prefrontal cortex of the human brain, there were no significant differences in the levels of expression between the cortices of normal subjects and those with major mental illnesses. The Western blot analysis confirmed the specificity of the antibodies. Thy-1 immunoblots revealed a single protein band at 25 kDa, corresponding to the molecular weight of human Thy-1 previously reported between

Figure 6 Intensity of L1 immunoreactivity in human prefrontal cortex as a function of cortical layers. The intensity was not different between diagnostic groups (P = 0.89).

23.5 and 29 kDa.29–31 The L1 immunoblots revealed two protein bands at 200 and 140 kDa. In the human brain L1 has a molecular weight of 200 kDa and can be cleaved to an extracellular amino acid fragment of 140 kDa.32,33 The present findings confirm those of previous studies that have reported that Thy-1 is preferentially located in areas of gray matter in the adult human brain.18–20 However, neither neuronal cell bodies nor glial cells are immunoreactive for Thy-1 in the human brain. Thus, within the gray matter, it is only the axons that are immunoreactive. As the axon fascicles enter the cortical gray matter they become intensely immunoreactive. The axon fascicles radiate across the cortex and display a granular pattern of staining that delineates the neurons in the superficial layers. In the developing human neocortex the amount of Thy-1 antigen expressed is barely detectable at 31 weeks of gestation but then increases rapidly to adult levels by 8 months of age.19,20 Thy-1 staining initially appears as fine granular staining of fibers evenly distributed throughout all layers. During the postnatal period fluorescent bands of axon fascicles form in the gray matter as described in this study. Tiveron et al34 demonstrated that Thy-1 expression by cells in vitro can inhibit the outgrowth of neurites and proposed that Thy-1 may be involved in the stabilization of neuronal connections. The timing of Thy-1 expression on axons in the human neocortex could well correspond to the stabilization of connections. However, it appears that the function of Thy-1 may vary depending on the demands of the system at particular stages, since during the development of the rodent brain, Thy-1 is expressed on various subsets of neuronal somas, dendrites and axons.35–39 L1 is expressed in the neuropil of the adult human frontal cortex and on fine fibers in the adjacent white matter. Tsuru et al,40 described ‘mild’ L1 staining in the cortex of a 14-year-old human but, in contrast to our results, staining was negative in the adjacent white matter. This discrepancy may be related to the age of the subject, to the different cortical areas studied, or to differences in antibodies or immunostaining procedures. L1 has been identified in the early axon fiber tracts in the developing CNS of rodents,41–43 but is not expressed after myelination.44,45 L1 is, however, retained on some unmyelinated axons in the adult.46–49 In such cases L1 is presumed to be involved in the maintenance of axon–axon contact in fascicles. Thus the fiber staining we describe may be located on a subset of unmyelinated fibers in the frontal cortex and white matter. Since L1 is not a significant component of serum proteins,10 it is presumed that the 50% reduction in the 140-kDa form of L1 in the CSF of patients with schizophrenia10 must reflect abnormal L1 function somewhere in the brain. The fact that we did not find a reduction in the level of L1 expression in the frontal cortex of people with schizophrenia is therefore somewhat surprising. In addition, Vawter et al,12 have not observed changes in L1 concentrations in post-mortem


specimens of hippocampus from patients with schizophrenia. It is, however, possible that other brain regions may be more involved. Alternatively, decreases in L1 concentrations may be related to changes in L1 turnover or degradation rates may be due to, for example, decreased activity of a proteolytic enzyme. Finally, since our antibody recognized both the 200and 140-kDa forms of L1, staining from the 200-kDa form may be masking any differences in the expression of the 140-kDa form. Acknowledgements Postmortem brain samples were provided by the Stanley Foundation Brain Consortium courtesy of Drs Llewellyn B Bigelow, Juraj Cervenak, Mary M Herman, Thomas M Hyde, Joel E Kleinman, Jose D Paltan, Robert M Post, E Fuller Torrey, Maree J Webster, and Robert H Yolken. Dr John J Hemperly (Becton Dickinson, Research Triangle Park, NC, USA) kindly provided the monoclonal L1 antibody and recombinant human L1GST fusion protein.

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