Specific regional transcription of apolipoprotein E in human brain neurons

Abstract

In central nervous system injury and disease, apolipoprotein E (APOE, gene; apoE, protein) might be involved in neuronal injury and death indirectly through extracellular effects and/or more directly through intracellular effects on neuronal metabolism. Although intracellular effects could clearly be mediated by neuronal uptake of extracellular apoE, recent experiments in injury models in normal rodents and in mice transgenic for the human APOE gene suggest the additional possibility of intraneuronal synthesis. To examine whether APOE might be synthesized by human neurons, we performed in situ hybridization on paraffin-embedded and frozen brain sections from three nondemented controls and five Alzheimer's disease (AD) patients using digoxigenin-labeled antisense and sense cRNA probes to human APOE. Using the antisense APOE probes, we found the expected strong hybridization signal in glial cells as well as a generally fainter signal in selected neurons in cerebral cortex and hippocampus. In hippocampus, many APOE mRNA-containing neurons were observed in sectors CA1 to CA4 and the granule cell layer of the dentate gyrus. In these regions, APOE mRNA containing neurons could be observed adjacent to nonhybridizing neurons of the same cell class. APOE mRNA transcription in neurons is regionally specific. In cerebellar cortex, APOE mRNA was seen only in Bergmann glial cells and scattered astrocytes but not in Purkinje cells or granule cell neurons. ApoE immunocytochemical localization in semi-adjacent sections supported the selectivity of APOE transcription. These results demonstrate the expected result that APOE mRNA is transcribed and expressed in glial cells in human brain. The important new finding is that APOE mRNA is also transcribed and expressed in many neurons in frontal cortex and human hippocampus but not in neurons of cerebellar cortex from the same brains. This regionally specific human APOE gene expression suggests that synthesis of apoE might play a role in regional vulnerability of neurons in AD. These results also provide a direct anatomical context for hypotheses proposing a role for apoE isoforms on neuronal cytoskeletal stability and metabolism.

Figure 1.

Specificity of DIG-labeled cRNA probes for human APOE. A: Southern blotting demonstrates that both antisense and sense APOE DIG-cRNA probes prepared from APOE3 cDNA hybridize specifically with 1163-bp APOE cDNA fragments (illustrated for APOE3 cDNA and APOE2 cDNA fragments; APOE4 not shown). B: Both antisense and sense APOE DIG-cRNA probes were able to detect 1.0 to 10 ng of APOE2 plasmid cDNA in slot blot analysis. C: Northern blot analysis (15 μg of total brain RNA loaded onto each lane) showed that only the antisense APOE DIG-cRNA probe (left) had specific hybridization with human APOE mRNA extracted from mouse brains of human APOE transgenic lines allele specific for APOE3 (E3–437), APOE2 (E2–205), and APOE4 (E4–81) and human frontal cortex (labeled Human). No mRNA signal was detected in the APOE knockout (KO (−/−)) mouse using antisense DIG-cRNA probe. Parallel blot hybridized with sense probe (right) did not yield any signal.

Figure 2.

In situ hybridization for APOE mRNA in frozen human liver sections of nondemented control (case 1) showing relative lack of signal with sense APOE DIG-cRNA probe (A) compared with strong signal in hepatocytes with antisense probe (B). s, sinusoid spaces with unstained red blood cells; cords of hepatocytes are indicated by arrows. Presence or absence of signal for Kupffer cells in B could not be determined due to strong hepatocyte signal. Bar, 20 μm.

Figure 3.

In situ hybridization for APOE mRNA in glial cells of cerebellar cortex corresponds to apoE immunolocalization. A: Relative lack of hybridization signal with sense APOE DIG-cRNA probes in paraffin sections of cerebellar cortex of AD patient (case 5). B: Strong hybridization signal along Purkinje cell/Bergmann glial cell layer with antisense probe for APOE mRNA in semi-adjacent sections of case 5 represents Bergmann glial cells (see below). C: Restriction of APOE mRNA hybridization pattern to glial cells in cerebellum was also observed clearly in frozen sections of nondemented control with ALS (case 1). D: Higher magnification of area demarcated above in B demonstrates signal in radial glial fibers (RGF) and Bergmann glial cell bodies (arrows) and lack of any signal in Purkinje cells (P). E: Immunocytochemical localization of apoE demonstrates presence of apoE immunoreactivity in similar distribution to mRNA localization, although Bergmann glial cell bodies and entire extent of radial glial fiber are more clearly seen. The faint background staining of Purkinje cell (P) shows relationship of unseen Purkinje cells to Bergmann glial cells in A to D. Bar, 60 μm (A and B) and 20 μm (C to E).

Figure 4.

In situ hybridization for APOE mRNA in glial cells and neurons of cerebral cortex corresponds to apoE immunolocalization. Hybridization with sense APOE DIG-cRNA probe results in some faint cell staining (arrows indicate background signal in neurons) in paraffin section from frontal cortex of AD patient (case 4; A) and frozen section from temporal lobe of nondemented control with ALS (case 1; D). B: Field from parallel processed paraffin section of frontal cortex of AD patient (case 4) hybridized with antisense APOE DIG-cRNA probe showing APOE mRNA-positive neurons (arrow) and an example of satellite glial cell (arrowhead). C: Hybridization signal was observed in scattered cells in paraffin section of frontal cortex from AD patient (case 5). Size and morphology of these cells was consistent with pyramidal cortical neurons (arrows). The eccentric location of signal in some cases suggests possible additional staining of satellite glial cells (arrowheads). E: Particularly distinctive hybridization signal was observed in frozen section of temporal lobe of nondemented control with ALS (case 1), whose parallel sections processed with sense probe showed low background sense probe signal (see D). Several APOE mRNA-positive neurons (arrows) and presumptive astrocytes (arrowheads) are indicated. F: Immunocytochemical localization of apoE in the parallel paraffin section of frontal cortex from AD patient (case 5; compared with in situ hybridization in C) demonstrated immunoreactive apoE in many neurons (arrows), glial cells (arrowheads), and senile plaques (SP). Bar, 20 μm.

Figure 5.

Paraffin sections of hippocampus from nondemented control (case 3) showed consistent pattern of apoE immunoreactivity (A) and in situ hybridization signal (B) for neuronal APOE translation and transcription in granule cell layer of dentate gyrus. Scattered apoE-immunoreactive neurons (arrows in A) and APOE mRNA-positive neurons (arrows in B) are located within the granule cell layer. From both immunolocalization and mRNA localization results, there are also clearly many granule cell neurons that do not contain signal. Staining of smaller non-neuronal cells (arrowheads) may variably represent astrocytes (arrowhead 1 in A), microglial cells (arrowhead 2 in A and arrowhead 2 in B), and satellite glial cells (arrowhead 1 in B). Bar, 20 μm.

Figure 6.

In situ hybridization demonstrated APOE mRNA signal in numerous neurons in CA1–2 (B), CA3 (C to E), and CA4 (F to H) sectors of human hippocampus. A: In situ hybridization with sense APOE DIG-cRNA probe hybridization with paraffin section of hippocampus of AD patient (case 8) showed faint background signal in some neurons (arrow) in CA1–2 sector. B: Hybridization with antisense APOE DIG-cRNA probe in parallel processed paraffin section of same region showed numerous APOE mRNA-positive neurons (arrows). In some cases, eccentric signal close to neurons (arrowhead) suggested signal in satellite glial cells. Comparison of apoE immunolocalization and APOE mRNA hybridization pattern of CA3 sector of another AD patient (case 5) demonstrated similar distribution of apoE immunoreactivity (C) and APOE mRNA hybridization signal (D) in neurons (arrows) and glial cells (arrowheads). E: APOE mRNA hybridization signal in paraffin-embedded section of CA3 sector in another AD patient (case 6) showed examples of APOE mRNA-negative or low-signal neurons (arrow 1), medium-signal, presumably positive neurons (arrow 2), and strong-signal neurons (arrow 3). Nearby are some presumptive glial cells (arrowheads.) with strong hybridization signal. F: ApoE immunoreactivity pattern in paraffin section of CA4 sector of nondemented control (case 2) showing immunoreactive neurons (arrow) and glial cells (arrowhead). G: APOE mRNA hybridization signal in paraffin section of CA4 sector of AD patient (case 4) showing numerous positive neurons (arrows) and glial cells (arrowheads). H: APOE mRNA hybridization signal in frozen section of CA4 sector of nondemented control with ALS (case 1) showing APOE mRNA-positive neurons (arrows) and glial cells (presumptive astrocytes, arrowhead). Bar, 20 μm.