Cannabinoid CB2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaque-associated glia in Alzheimer's disease brains

Abstract

The endocannabinoid system is still poorly understood. Recently, the basic elements that constitute it, i.e., membrane receptors, endogenous ligands, and mechanisms for termination of the signaling process, have been partially characterized. There is a considerable lack of information, however, concerning the distribution, concentration, and function of those components in the human body, particularly during pathological events. We have studied the status of some of the components of the endocannabinoid system, fatty acid amide hydrolase and cannabinoid CB1 and CB2 receptors, in postmortem brains from patients with Alzheimer's disease. Using specific polyclonal antibodies, we have performed immunohistochemical analysis in hippocampus and entorhinal cortex sections from brains of Alzheimer's disease patients. Our results show that both fatty acid amide hydrolase and cannabinoid CB2 receptors are abundantly and selectively expressed in neuritic plaque-associated astrocytes and microglia, respectively, whereas the expression of CB1 receptors remains unchanged. In addition, the hydrolase activity seems to be elevated in the plaques and surrounding areas. Thus, some elements of the endocannabinoid system may be postulated as possible modulators of the inflammatory response associated with this neurodegenerative process and as possible targets for new therapeutic approaches.

Figure 1.

Western blots of CB₁ (lanes 1, 2), FAAH (lanes 3, 4), and CB₂ (lanes 5, 6) immunoreactivities in human temporal cortex from an AD patient. Single bands of ∼50 kDa (CB₁ and FAAH) or 60 kDa (CB₂) were observed (lanes 1, 3, and 5, respectively). No immunoreactivities were detected when the primary antibodies were preincubated with the respective immunizing peptides (lanes 2, 4, and 6).

Figure 2.

A-F, FAAH (A-C, E) and GFAP (D) immunoreactivities in parahippocampal cortex and FAAH activity in neuritic plaques (F). A, FAAH staining in a healthy individual sample. Note the neuronal pattern of staining (inset). B, Low and high (inset) magnifications of FAAH immunoreactivity in parahippocampal cortex of an AD case. Note the intense signal for FAAH in hypertrophied astrocytes surrounding neuritic plaques. C, Detail of FAAH immunoreactivity in hypertrophied astrocytes. D, Low and high (inset) magnification of GFAP immunoreactivity in an AD case. Note that the signal is detectable in both protoplasmic and fibrous (arrow) astrocytes. E, FAAH staining after preabsorption and coincubation of the antibody with the immunizing peptide. Note the absence of any detectable signal. F, FAAH activity. Individual plaques were dissected under microscope, homogenized, and assayed for FAAH activity using the conversion of [¹⁴C]AEA to [¹⁴C]ethanolamine during a 15 min incubation. Shown is the mean of five individual determinations; groups were significantly different using unpaired t tests with p < 0.05. Scale bars: A, B, D, 800 μm; E, inset in A, 400 μm; insets in B and D, 200 μm; C, 100 μm.

Figure 3.

CB₂ (A-C, E) and CD68 (D) stainings in parahippocampal cortex. A, CB₂ staining in a healthy individual sample. No detectable signal could be seen. B, Low and high (inset) magnifications of CB₂ immunoreactivity in parahippocampal cortex of an AD case. Note the intense signal for CB₂ in microglial cells located on neuritic plaques. C, Detail of CB₂ immunoreactivity in neuritic plaque-associated microglia. D, Low and high (inset) magnification of CD68 immunoreactivity in an AD case. E, CB₂ staining after preabsorption and coincubation of the antibody with the immunizing peptide. Note the absence of any detectable signal. Scale bars: A, B, D, E, 800 μm; insets in B and D, 200 μm; C, 100 μm.

Figure 4.

FAAH and CB₂ are expressed in glial cells associated with β-amyloid-enriched neuritic plaques. A, B, FAAH (brown) and β-amyloid peptide (blue) stainings. Note that FAAH-positive cells are astrocytes surrounding β-amyloid-enriched plaques. C, D, CB₂ (brown) and β-amyloid peptide (blue) stainings. CB₂ immunostaining is limited to plaque-associated microglial cells.

Figure 5.

CB₁ staining in parahippocampal cortex. A, CB₁ staining in a healthy individual sample. Pyramidal cortical cells showed moderate to intense staining level (inset). B, Low and high (inset) magnifications of CB₁ immunoreactivity in parahippocampal cortex of an AD case. Note the general lower intensity of the signal for CB₁ and how the neuritic plaques can be observed easily (arrows). C, Detail of CB₁ immunoreactivity, showing no changes in the vicinity of neuritic plaques (arrows). D, CB₁ staining after preabsorption and coincubation of the antibody with the immunizing peptide. Note the absence of any detectable signal. Scale bars: A, B, D, 800 μm; inset in B, 400 μm; inset in A, 200 μm; C, 100 μm.