Multiple sclerosis (MS) is an autoimmune disease in which myelin is progressively degraded. Because degraded myelin may both initiate and accelerate disease progression, clearing degraded myelin from extracellular spaces may be critical. In this study, we prepared myelin vesicles (MV) from rat brains as a model of degraded myelin. Murine embryonic fibroblasts (MEFs) rapidly internalized MVs, which accumulated in lysosomes only when these cells expressed low-density lipoprotein receptor-related protein (LRP1). Receptor-associated protein (RAP), which binds LRP1 and inhibits interaction with other ligands, blocked MV uptake by LRP1-expressing MEFs. As a complementary approach, we prepared primary cultures of rat astrocytes, microglia and oligodendrocytes. All three cell types expressed LRP1 and mediated MV uptake, which was inhibited by RAP. LRP1 gene-silencing in oligodendrocytes also blocked MV uptake. Myelin basic protein (MBP), which was expressed as a recombinant protein, bound directly to LRP1. MBP-specific antibody inhibited MV uptake by oligodendrocytes. In experimental autoimmune encephalomyelitis in mice, LRP1 protein expression was substantially increased in the cerebellum and spinal cord. LRP1 colocalized with multiple CNS cell types. These studies establish LRP1 as a major receptor for phagocytosis of degraded myelin, which may function alone or in concert with co-receptors previously implicated in myelin phagocytosis.

Normal brain function is highly dependent on oxygen and nutrient supply and when the demand for oxygen exceeds its supply, hypoxia is induced. Acute episodes of hypoxia may cause a depression in synaptic activity in many brain regions, whilst prolonged exposure to hypoxia leads to neuronal cell loss and death. Acute inadequate oxygen supply may cause anaerobic metabolism and increased respiration in an attempt to increase oxygen intake whilst chronic hypoxia may give rise to angiogenesis and erythropoiesis in order to promote oxygen delivery to peripheral tissues. The effects of hypoxia on neuronal tissue are exacerbated by the release of many inflammatory agents from glia and neuronal cells. Cytokines, such as TNF-α, and IL-1β are known to be released during the early stages of hypoxia, causing either local or systemic inflammation, which can result in cell death. Another growing body of evidence suggests that inflammation can result in neuroprotection, such as preconditioning to cerebral ischemia, causing ischemic tolerance. In the following review we discuss the effects of acute and chronic hypoxia and the release of pro-inflammatory cytokines on synaptic transmission and plasticity in the central nervous system. Specifically we discuss the effects of the pro-inflammatory agent TNF-α during a hypoxic event.

␣ 2 -Macroglobulin (␣ 2 M) is a plasma protease inhibitor, which reversibly binds growth factors and, in its activated form, binds to low density lipoprotein receptor-related protein (LRP-1), an endocytic receptor with cell signaling activity. Because distinct domains in ␣ 2 M are responsible for its various functions, we hypothesized that the overall effects of ␣ 2 M on cell physiology reflect the integrated activities of multiple domains, some of which may be antagonistic. To test this hypothesis, we expressed the growth factor carrier site and the LRP-1 recognition domain (RBD) as separate GST fusion proteins (FP3 and FP6, respectively). 2 is a 718-kDa homotetrameric glycoprotein, found in the plasma and extracellular spaces, which was first recognized as a broad spectrum protease inhibitor (1). Reaction with proteases induces a major conformational change in ␣ 2 M so that the protease is physically trapped (2, 3). The same conformational change reveals a cryptic recognition site for low density lipoprotein receptor-related protein (LRP-1) (4, 5). Because of the function of LRP-1 as an endocytic receptor, ␣ 2 M-protease complexes are rapidly cleared from the bloodstream and probably other sites of generation (6).In addition to its role as a protease inhibitor, ␣ 2 M is an important carrier of specific growth factors, including transforming growth factor-␤ (TGF-␤), platelet-derived growth factor-BB (PDGF-BB), nerve growth factor-␤ (NGF-␤), and neurotrophin-4 (7, 8). ␣ 2 M-carrier interactions are principally reversible in nature (7). As a result, ␣ 2 M may inhibit growth factor activity (9, 10) or stabilize the growth factor for potential delivery to cell signaling receptors (11). There also is evidence that ␣ 2 M, which is "activated" by reaction with proteases, initiates cell signaling by binding to LRP-1 (12-15) or other receptors, such as glucose-regulated protein-78 (Grp 78) (16,17). However, in studies with intact ␣ 2 M, the possibility that cell signaling results from growth factors that are carried by ␣ 2 M must be considered (11,18).A structural model of ␣ 2 M has been developed, based on the crystal structure of complement component C3, which is homologous to ␣ 2 M (19). This model describes ␣ 2 M as a modular structure, consisting of multiple independently folded domains. To localize ␣ 2 M domains that are responsible for various activities, our laboratory generated a library of glutathione S-transferase (GST) fusion proteins, containing overlapping segments of the ␣ 2 M subunit (20 -22). Fusion protein-3 (FP3) includes residues 591-774 and contains the sequence in ␣ 2 M responsible for binding growth factors. FP6 contains amino acids 1242-1451 and the LRP-1-binding site, in which a single ␣-helix that includes Lys 1370 and Lys 1374 plays a central role (23,24). Assignment of ␣ 2 M activities to specific fusion proteins, such as FP3 and FP6, has been validated by mutagenesis of full-length recombinant ␣ 2 M (25, 26). Thus, the ␣ 2 M fusion proteins provide an opportunity to assess activities assigne...

An inadequate supply of oxygen in the brain may lead to the introduction of an inflammatory response through neuronal and glial cells that can result in neuronal damage. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that is released during acute hypoxia and can have neurotoxic or neuroprotective effects in the brain. Both TNF-α and interleukin-1β (IL-1β) have been shown by a number of research groups to alter synaptic scaling and also to inhibit long-term potentiation (LTP) in the hippocampus when induced by specific high frequency stimulation protocols. In this study we have examined the effects of TNF-α on synaptic transmission and plasticity in hippocampal slices after acute hypoxia using two high frequency stimulation protocols. Field excitatory postsynaptic potentials were elicited in the medial perforant pathway of the dentate gyrus. Exogenous TNF-α (5 ng/ml) attenuated LTP induced by theta burst stimulation but had no effect on LTP induced by a more prolonged high frequency stimulation (HFS). Pre-treatment with lipopolysaccharide (100 ng/ml) or TNF-α but not IL-1β (4 ng/ml) prior to a 30 min hypoxic insult resulted in a significant enhancement of LTP post hypoxia when induced by the HFS. Anti-TNF, 3,6 dithiothalidomide (a TNF-α synthesis inhibitor) and SB203580 (a p38 MAPK inhibitor) significantly reduced this effect. These results indicate an important modulatory role for elevated TNF-α levels on LTP in the hippocampus after an acute hypoxic event.

In the CNS short episodes of acute hypoxia can result in a decrease in synaptic transmission which may be fully reversible upon re-oxygenation. Stabilization of hypoxiainducible factor (HIF) by inhibition of prolyl hydroxylase domain (PHD) enzymes has been shown to regulate the cellular response to hypoxia and confer neuroprotection both in vivo and in vitro. Hypoxic preconditioning has become a novel therapeutic target to induce neuroprotection during hypoxic insults. However, there is little understanding of the effects of repeated hypoxic insults or pharmacological PHD inhibition on synaptic signalling. In this study we have assessed the effects of hypoxic exposure and PHD inhibition on synaptic transmission in the rat CA1 hippocampus. Field excitatory postsynaptic potentials (fEPSPs) were elicited by stimulation of the Schaffer collatoral pathway. 30 min hypoxia (gas mixture 95% N2/5% CO2) resulted in a significant and fully reversible decrease in fEPSP slope associated with decreases in partial pressures of tissue oxygen. 15-30 min of hypoxia was sufficient to induce stabilization of HIF in hippocampal slices. Exposure to a second hypoxic insult after 60 min resulted in a similar depression of fEPSP slope but with a significantly greater rate of recovery of the fEPSP. Prior single treatment of slices with the PHD inhibitor, dimethyloxalylglycine (DMOG) also resulted in a significantly greater rate of recovery of fEPSP post hypoxia. These results suggest that hypoxia and 'pseudohypoxia' preconditioning may improve the rate of recovery of hippocampal neurons to a subsequent acute hypoxia.