AD is the most common cause of dementia in the elderly and is characterized by parenchymal deposits of fibrillar amyloid-β (fAβ) surrounded by microglia and astrocytes. In culture, these and other SR-bearing cells from the CNS interact with fAβ which results in activation of nuclear factor-κB (NF-κB) and its binding to DNA, increases in protein-tyrosine phosphorylation, and secretion of pro-inflammatory substances, e.g., reactive oxygen species (ROS), TNF-α, and complement components by these cells (McDonald et al., 1998; Combs et al., 1999, 2001; Terry, 1999; Nakai et al., 2001). SR-A, SR-BI, and CD36 have all been suggested to participate in the pathogenesis of AD.
SR-A is upregulated in a subset of reactive microglia in lesions of ischemia and in microglia associated with amyloid deposits in brains of patients with AD (Christie et al., 1996; Honda et al., 1998). While these investigators differ with respect to expression of SR-A by Mato cells and by microglia in normal human brain, they agree that SR-A is strongly expressed by microglia in AD brain. Similar observations were made in brains from APP23 mice, a transgenic mouse model for AD, where SR-A is localized on microglia associated with parenchymal and vascular amyloid deposits (Bornemann et al., 2001). These observations suggest induction of SR-A on microglia in pathological conditions. Whether upregulation of SR-A in AD brain results from direct microglial interaction with fAβ, and/or activation of these cells by cytokines/chemokines present in AD lesions are important areas to be investigated.
SR-A was the first receptor shown to participate in binding and internalization of fAβ 1–42 by cultured murine microglia and other mononuclear phagocytes (El Khoury et al., 1996; Paresce et al., 1996). Subsequently, we and others reported that neonatal microglia from SR-A−/− mice bound ∼50% less fluorescently tagged fAβ 1–42 than wild-type microglia (Chung et al., 2001; Husemann et al., 2001a). These findings, and the observation that microglia associated with Aβ deposits in AD brain express SR-A, suggested a role for this receptor in the pathogenesis of AD. However, Huang et al. (1999) found that transgenic mice lacking SR-A (SR-A−/−) and overexpressing human amyloid precursor protein (APP) in their brains (SR-A−/−/PDAPP mice) exhibited AD-like brain pathology similar to that observed in SR-A+/+/PDAPP mice. These investigators concluded that interaction between SR-A and fAβ is not required for the development of AD-like pathology in mice. However, their results do not rule out a role for microglia in AD. We have found that neonatal microglia from SR-A−/− mice adhere to matrix-associated fAβ, and secrete ROS in response to fAβ as efficiently as wild-type microglia. These findings suggest that cell receptors other than SR-A mediate Aβ-dependent ROS secretion by these cells in adult mice (Husemann et al., 2001a). Whether SR-A-fAβ interaction leads to the production of pro-inflammatory substances other than ROS by microglia remains to be examined.
Coraci et al. (2002) have implicated CD36 in the pathogenesis of AD. These investigators detected low levels of CD36 on microglia in normal adult brain and in brains of patients with AD, multiple sclerosis and Parkinson's disease but could not discriminate whether there were differences in CD36 expression in normal versus diseased brain. Furthermore, these authors identified CD36 as a receptor for fAβ 1–42 that participates in signaling ROS production by human monocyte-derived macrophages, human neonatal microglia, and N9 cells upon interaction with fAβ 1–42 in vitro. They found that murine antibodies directed against human CD36 (clones SMΦ and NL07) reduced by ∼50% secretion of ROS when these cells were plated on surfaces coated with fAβ 1–42 but only minimally affected cell adhesion to these surfaces. Although these authors observed a significant inhibitory effect of mouse antihuman CD36 antibodies on secretion of ROS upon interaction of N9 cells with fAβ 1–42, others reported that these antibodies do not bind to murine CD36 (Daviet et al., 1995; Navazo et al., 1996; Puente Navazo et al., 1996). Further studies are needed to determine the mechanism(s) by which these antibodies inhibit ROS secretion by mouse and human mononuclear phagocytes (Maxeiner et al, 1998; Coraci et al., 2002).
We have used CD36−/− microglia to further explore the role(s) of CD36 in fAβ 1–42-stimulated ROS production. Since adhesion of microglia to plastic or glass stimulates them to produce ROS, we used surfaces coated with collagen IV, a matrix protein to which microglia adhere weakly, if at all, to measure basal ROS release by these cells (Husemann et al., 2001a). We compared H2O2 production by wild-type and CD36−/− murine neonatal microglia plated on surfaces coated with collagen IV alone with surfaces coated with collagen IV and fAβ, or plated on collagen IV and stimulated with phorbol myristate acetate (PMA), a protein kinase C-activator known to signal maximal H2O2 production by mononuclear phagocytes (Table 2). The results of these experiments were informative and surprising. First, CD36−/− microglia released 68% more H2O2 than wild-type microglia when plated on collagen IV alone. Second, there was no significant difference in H2O2 production by CD36−/− microglia plated on surfaces coated with collagen IV alone than on surfaces coated with collagen IV and fAβ. As expected, microglia from wild-type mice produced 23% more H2O2 when plated on surfaces coated with collagen IV and fAβ than on surfaces coated with collagen IV alone (Coraci et al., 2002; Husemann et al., 2001a). Third, PMA stimulated a 57% increase in H2O2 production by wild-type microglia but no significant increase in H2O2 production by CD36−/− microglia. These experiments indicate that, at least with respect to H2O2 production, neonatal microglia from CD36−/− mice are constitutively “activated.” They suggest that in wild-type microglia CD36 exerts a negative regulatory effect on protein kinase C and/or other enzymes that regulate H2O2 production. Therefore, it seems likely that CD36−/− mice will be less helpful than hypothesized in dissecting the role(s) of CD36 in signaling ROS production stimulated by fAβ 1–42 in wild-type microglia. Finally, these experiments emphasize the need for caution in ascribing changes in phenotype and/or physiological functions to specific receptors and biochemical pathways in knockout mice. In the case of CD36 deficient microglia, absence of CD36 appears to correlate with a constitutive gain of function.
Table 2. H2O2 production (in pmoles/150,000 cells) by neonatal microglia from wild-type or CD36−/− mice under unstimulated conditions and in response to fAβ 1–42 or PMA†
| ||Without stimulus||+ fAβ 1–42||+PMA|
|Wild-type microglia||422 ± 20||519 ± 19 (Δ: 97 ± 25; P ≤ 0.05*)||663 ± 63 (Δ: 241 ± 67; P ≤ 0.05*)|
|CD36−/− microglia||711 ± 53; P ≤ 0.005*)||731 ± 48 (Δ: 21 ± 12; n.s.**)||801 ± 83 (Δ: 91 ± 42; n.s.**)|
SR-BI participates in binding and endocytosis of fAβ 1–42 by adult astrocytes and cerebrovascular smooth muscle cells in vitro (Prior et al., 2000; Husemann, Wyss-Coray et al., submitted). The role of SR-BI in Aβ clearance by these cells in vivo in normal and AD brain remains to be determined. Whether interaction of SR-BI with fAβ 1–42 activates signal transduction pathways in astrocytes and cerebrovascular smooth muscle cells, as it does with HDL in endothelium (Yuhanna et al., 2001), is unknown.
Increased production of human Aβ in the brains of adult mice expressing a transgene encoding this protein resulted in AD-like pathology, synaptic transmission deficits, and behavioral impairments. Immunization of these mice with human fAβ resulted in marked reductions in the Aβ burdens in the brains of these mice and protected them from the age-related behavioral impairments seen in un-immunized littermates (Schenk et al., 1999; Morgan et al., 2000). Several mechanisms have been suggested as mediating these effects (Bard et al., 2000; DeMattos et al., 2001). To these should be added the possibility that antibodies versus fAβ increase the accessibility of this protein to SR-bearing CNS cells, facilitating the removal of this protein.
Diabetic Peripheral Neuropathy
Glucose-modified proteins, called advanced glycation end products (AGEs) accumulate on long-lived proteins such as myelin and extracellular matrix proteins in patients with diabetes (Brownlee, 1992). These AGE-induced changes can affect cell adhesion, growth, and matrix accumulation. SR-A, SR-BI, and CD36 all serve as receptors for AGEs (El Khoury et al., 1994; Ohgami et al., 2001a, b). AGE-modified proteins also alter vascular function by interacting with specific receptors on macrophages and endothelial cells, inducing changes that are associated with matrix overproduction, focal thrombosis, and vasoconstriction (Baynes, 2001). Thus, interactions of SR with AGEs may contribute to the development of diabetic peripheral neuropathy.