Increased 5-lipoxygenase immunoreactivity in the hippocampus of patients with Alzheimer's disease

Abstract

The proinflammatory enzyme 5-lipoxygenase (5-LOX) is upregulated in Alzheimer's disease (AD), but its localization and association with the hallmark lesions of the disease, beta-amyloid (Abeta) plaques and neurofibrillary tangles (NFTs), is unknown. This study examined the distribution and cellular localization of 5-LOX in the medial temporal lobe from AD and control subjects. The spatial relationship between 5-LOX immunoreactive structures and AD lesions was also examined. We report that, in AD subjects, 5-LOX immunoreactivity is elevated relative to controls, and its localization is dependent on the antibody-targeted portion of the 5-LOX amino acid sequence. Carboxy terminus-directed antibodies detected 5-LOX in glial cells and neurons, but less frequently in neurons with dystrophic (NFT) morphology. In contrast, immunoreactivity observed using 5-LOX amino terminus-directed antibodies was virtually absent in neurons and abundant in NFTs, neuritic plaques, and glia. Double-labeling studies showed a close association of 5-LOX-immunoreactive processes and glial cells with Abeta immunoreactive plaques and vasculature and also detected 5-LOX in tau immunoreactive and amyloid containing NFTs. Different immunolabeling patterns with antibodies against carboxy vs amino terminus of 5-LOX may be caused by post-translational modifications of 5-LOX protein in Abeta plaques and NFTs. The relationship between elevated intracellular 5-LOX and hallmark AD pathological lesions provides further evidence that neuroinflammatory pathways contribute to the pathogenesis of AD.

Figure 1

Schematic illustration of representative distributions of 5-lipoxygenase (5-LOX)-labeled elements, using the 5-LOX antibodies shown in Table 1, in the hippocampus of an AD subject.

Figure 2

IHC detection of 5-LOX protein in the hippocampus of control individuals with no cognitive impairment (A,D) and patients with Alzheimer's disease (AD) (B,C,E,F) using antisera recognizing the NH₂ terminus (A–C; Santa Cruz-N) and aa 130–149 (D–F; Cayman) of 5-LOX. With either antibody, there is a very low level of immunoreactivity in the subiculum/CA1 from a control case (A,D). In the CA1 (B,E) and subiculum (C,F) of a representative AD case, neurons with twisted apical dendrites and “tangled” material in cell bodies are strongly immunoreactive with either the aa 130–149 (E,F)– or NH₂ terminus (B,C)–specific antibodies. 5-LOX immunoreactive tangled neuritic structures are also seen in the neuropil (F, arrow). Most 5-LOX–immunostained neurons in subiculum resemble neurofibrillary tangles (NFTs) (F). Bar = 25 μm.

Figure 3

5-LOX immunoreactivity in the dentate gyrus (A), CA3 hippocampus (B), and temporal cortex (C,D) from an AD patient, using the antiserum recognizing 5-LOX aa 130–149 (Cayman). Accumulations of 5-LOX–immunoreactive (ir) plaque-like clumps of cells and cell processes are evident in all three regions. Bar = 50 μm.

Figure 4

A comparison of IHC detection of 5-LOX protein in the CA1 hippocampus of patients with AD using antisera recognizing the NH₂ terminus (A, RDI-2), aa 442–590 (B, Transduction), and COOH terminus (C, RDI-1; D, Santa Cruz-C). The NH₂ terminus antibody shows robustly stained cells with twisted apical dendrites (A), whereas the aa 442–590 antibody detects sparse tangle-looking cells (B). Both of the C terminus antibodies recognize neuronal and glial cells (C,D). Bar = 25 μm.

Figure 5

IHC study of 5-LOX protein expression in the AD hippocampus using antisera recognizing aa 130–149 (Cayman) of 5-LOX. Compared with the regular IHC protocol (A), preabsorbing the antisera with purified 5-LOX peptide (B) or omission of the primary antisera (C) reduced immunostaining signal to background levels. Western blot analysis (D) of 5-LOX protein expression in the hippocampus from AD patients (AD, three different individuals) and control subjects with no cognitive impairment (NCI, two different individuals), using the same (Cayman) antibody, shows a prominent 80-kDa band (matching the known molecular mass of BSA) in AD tissues, whereas this band is hardly visible in control hippocampi. Bar = 50 μm.

Figure 6

Dual chromogen IHC for 5-LOX (Cayman; black, DAB/nickel) and amyloid-beta (Aβ; red; Vector red) in the hippocampus (A,B), inferior temporal (C), and entorhinal cortex (D) from an AD patient. Numerous large astrocytes surround Aβ plaques (A,B) and Aβ-containing blood vessels (B) in the hippocampus. A subset of plaques colocalized both peptides in the temporal cortex (C). In the entorhinal cortex lamina II, double IHC shows intensely 5-LOX–immunoreactive NFTs codistributed with small Aβ aggregates (D). Bar: A,C = 50 μm; B,D = 25 μm.

Figure 7

Dual immunofluorescence for 5-LOX (Santa Cruz-N; red) and tau (green) in the hippocampus (A,B) from an AD patient. Solid arrows point to the same cell that is positive for 5-LOX and tau. Some of the 5-LOX-ir cells (A, empty arrow) do not contain tau accumulation. Combined 5-LOX IHC (C, DAB/nickel) and X-34 histochemistry (D, blue fluorescence) in the same entorhinal cortex tissue section show that, in advanced AD, all of the 5-LOX-ir lamina II neurons also contain pathological β-pleated sheet conformed protein aggregates. Bar: A,B = 25 μm; C,D = 50 μm.