A decrease of reelin expression as a putative vulnerability factor in schizophrenia

Abstract

Postmortem prefrontal cortices (PFC) (Brodmann's areas 10 and 46), temporal cortices (Brodmann's area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (approximately 50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from gamma-aminobutyric acid (GABA)A receptors alpha1 and alpha5 and nicotinic acetylcholine receptor alpha7 subunits. Whereas the expression of the alpha7 nicotinic acetylcholine receptor subunit was normal, that of the alpha1 and alpha5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of approximately 50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability "two-hit" model for the etiology of schizophrenia.

Figure 1

In PFC of schizophrenia patients (SP), the decrease in RELN mRNA is paralleled by an increase of GABA_A α₁ and α₅ and no change in nACh α₇-receptor subunit mRNA. ∗, P < 0.01; ∗∗, P < 0.02; ∗∗∗, P < 0.05; NS, not significant; SP vs. nonpsychiatric subjects (NPS); Student’s t test. NPS: = 19, ▿ = 20, □ = 22, ○ = 23, ⋄ = 24, ▵ = 25, ⌓ = 26; SP: ▾ = 1, ▴ = 2, • = 4, = 5, ■ = 6, █ = 7, ♦ = 9, = 15.

Figure 2

Photomicrographs of 20-μm sections through human PFC immunolabeled for RELN (A) and DAB1 (B). The Inset in A is a higher magnification of the boxed area showing RELN-immunopositive interneurons. C shows double-labeling with DAB1 (blue-gray) and neuronal nuclei-specific protein [NeuN (40)] antisera, which labels most neurons (brown). Note that small cells (arrows) are immunonegative for the neuronal marker and that not all pyramidal cells are immunopositive for DAB1 (left pyramidal cell not double-labeled). (Bar = 150 μm for A and B and 20 μm for C and for the inset in A.) Western blotting of RELN (D) and DAB1 (E) in representative extracts from the temporal cortex of schizophrenia patients (SP) and nonpsychiatric subjects (NPS). Note that RELN ≈400-kDa immunoreactive bands (indicated by the arrow) have higher intensity in NPS (lanes 1, 2, and 3) than in SP (lanes 4, 5, and 6). In contrast, DAB1 ≈86-kDa bands (indicated by the arrow)are similar in the SP (lanes 10, 11, and 12) and NPS (lanes 7, 8, and 9). In D, 200, 400, and 1,000 μg of protein was resolved on 7.5% acrylamide gel electrophoresis using RELN 142 (1:500) and β-actin (1:5000) antiserum. In E, 25, 50, and 100 μg of protein was resolved on 7.5% acrylamide gel using DAB1 B3 antiserum (1:1000) and β-actin (1:50000) antiserum. Optical densities of the bands on the autoradiogram were quantified by using the Loats Image Analyses System (38).

Figure 3

Mean (±SE) of RELN-positive neurons in PFC layer I through VI of schizophrenia patients (patients 1, 2, 4, 5, 7, 8, 9) and nonpsychiatric subjects (subjects 10, 21, 22, 24, 25, 26). ∗, P < 0.01, Student’s t test.)