Migration of activated macrophages is essential for resolution of acute inflammation and the initiation of adaptive immunity. Here, we show that efficient macrophage migration in inflammatory environment depends on Mac-1 recognition of a binary complex consisting of fibrin within the provisional matrix and the protease tPA (tissue-type plasminogen activator). Subsequent neutralization of tPA by its inhibitor PAI-1 enhances binding of the integrinprotease-inhibitor complex to the endocytic receptor LRP (lipoprotein receptor-related protein), triggering a switch from cell adhesion to cell detachment. Genetic inactivation of Mac-1, tPA, PAI-1 or LRP but not the protease uPA abrogates macrophage migration. The defective macrophage migration in PAI-1-deficient mice can be restored by wild-type but not by a mutant PAI-1 that does not interact with LRP. In vitro analysis shows that tPA promotes Mac-1-mediated adhesion, whereas PAI-1 and LRP facilitate its transition to cell retraction. Our results emphasize the importance of ordered transitions both temporally and spatially between individual steps of cell migration, and support a model where efficient migration of inflammatory macrophages depends on cooperation of three physiologically prominent systems (integrins, coagulation and fibrinolysis, and endocytosis).

The amyloid precursor protein (APP) is cleaved to produce the Alzheimer disease-associated peptide A␤, but the normal functions of uncleaved APP in the brain are unknown. We found that APP was present in the postsynaptic density of central excitatory synapses and coimmunoprecipitated with N-methyl-Daspartate receptors (NMDARs). The presence of APP in the postsynaptic density was supported by the observation that NMDARs regulated trafficking and processing of APP; overexpression of the NR1 subunit increased surface levels of APP, whereas activation of NMDARs decreased surface APP and promoted production of A␤. We transfected APP or APP RNA interference into primary neurons and used electrophysiological techniques to explore the effects of APP on postsynaptic function. Reduction of APP decreased (and overexpression of APP increased) NMDAR whole cell current density and peak amplitude of spontaneous miniature excitatory postsynaptic currents. The increase in NMDAR current by APP was due to specific recruitment of additional NR2B-containing receptors. Consistent with these findings, immunohistochemical experiments demonstrated that APP increased the surface levels and decreased internalization of NR2B subunits. These results demonstrate a novel physiological role of postsynaptic APP in enhancing NMDAR function.Alzheimer disease (AD) 5 is an age-related neurodegenerative disease characterized by the progressive loss of synapses and neurons and by the formation of amyloid plaques and neurofibrillary tangles. Amyloid plaques are composed predominantly of the A␤ peptide, a 40-or 42-amino acid cleavage product of amyloid precursor protein (APP). APP is a transmembrane protein of unknown function that undergoes extracellular cleavage by one of two activities, ␣-or ␤-secretase, resulting in the formation of large N-terminal extracellular fragments of secreted APP and smaller, membrane-bound C-terminal fragments. If the initial cleavage event occurs via ␤-secretase, then subsequent cleavage of the C-terminal fragment by ␥-secretase results in the production of A␤ (1).Clues to APP function may be gleaned from studies of its different cleavage products or isoforms. Soluble forms of APP have been found to be neurotrophic, and some splice variants have proteinase inhibitor activity (2). The APP intracellular domain may alter gene transcription in conjunction with cytoplasmic proteins (3). In addition, the A␤ peptide has been shown to inhibit glutamate receptor activity (4, 5). However, the function of full-length APP is undefined, and it is likely that full-length APP performs distinct roles from any its cleavage products. One recent study showed that mice lacking APP have impaired development of neuromuscular junctions (6), suggesting an important role for APP in maintaining active synapses. Understanding APP function in central neurons may provide valuable information in generating interventions against the generation of A␤ and thus AD pathogenesis and its accompanying memory loss.Further evidence for a synaptic function of A...

Proteases contribute to a variety of processes in the brain; consequently, their activity is carefully regulated by protease inhibitors, such as neuroserpin. This inhibitor is thought to be secreted by axons at synaptic regions where it controls tissue-type plasminogen activator (tPA) activity. Mechanisms regulating neuroserpin are not known, and the current studies were undertaken to define the cellular pathways involved in neuroserpin catabolism. We found that both active neuroserpin and neuroserpin⅐tPA complexes were internalized by mouse cortical cultures and embryonic fibroblasts in a process mediated by the low density lipoprotein receptor-related protein (LRP). Surprisingly, despite the fact that active neuroserpin is internalized by LRP, this form of the molecule does not directly bind to LRP on its own, indicating the requirement of a cofactor for neuroserpin internalization. Our studies ruled out the possibility that endogenously produced plasminogen activators (i.e. tPA and urokinase-type plasminogen activator) are responsible for the LRP-mediated internalization of active neuroserpin, but could not rule out the possibility that another cell-associated proteases capable of binding active neuroserpin functions in this capacity. In summary, neuroserpin levels appear to be carefully regulated by LRP and an unidentified cofactor, and this pathway may be critical for maintaining the balance between proteases and inhibitors.

Dab1 is an intracellular adaptor protein that interacts with amyloid precursor protein (APP) and apoE receptor 2 (apoEr2), increases their levels on the cell surface, and increases their cleavage by ␣-secretases. To investigate the mechanism underlying these alterations in processing and trafficking of APP and apoEr2, we examined the effect of Fyn, an Src family-tyrosine kinase known to interact with and phosphorylate Dab1. Co-immunoprecipitation, co-immunostaining, and fluorescence lifetime imaging demonstrated an association between Fyn and APP. Fyn induced phosphorylation of APP at Tyr-757 of the 757 YENPTY 762 motif and increased cell surface expression of APP. Overexpression of Fyn alone did not alter levels of sAPP␣ or cytoplasmic C-terminal fragments, although it significantly decreased production of A␤. However, in the presence of Dab1, Fyn significantly increased sAPP␣ and C-terminal fragments. Fyn-induced APP phosphorylation and cell surface levels of APP were potentiated in the presence of Dab1. Fyn also induced phosphorylation of apoEr2 and increased its cell surface levels and, in the presence of Dab1, affected processing of its C-terminal fragment. In vivo studies showed that sAPP␣ was decreased in the Fyn knock-out, supporting a role for Fyn in APP processing. These data demonstrate that Fyn, due in part to its effects on Dab1, regulates the phosphorylation, trafficking, and processing of APP and apoEr2.

No abstract

The analysis of fluorescence lifetime imaging microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles.

The integrin Mac-1 plays a critical role in Fc receptor (FcR)-mediated antibody-dependent cellular cytotoxicity (ADCC). However, the mechanism by which Mac-1 facilitates the functions of FcγRIIA, a major FcR expressed on human leukocytes, is not fully understood. We report here that Mac-1 sustains cell adhesion, enhances cell spreading, and accelerates cell migration on pre-formed immune complexes (ICs) by directly interacting with FcγRIIA but not with the IC substrate. Coupling Mac-1 to FcγRIIA allows FcγRIIA to reside in the leading front of actin polymerization at the filopodial extension, and thus could potentially enhance the FcγRIIA-mediated cell spreading and migration. Direct interaction between Mac-1 and FcγRIIA is demonstrated by co-immunoprecipitation, by cell surface co-localization, and by solid-phase binding assays using recombinant α M I-domain and soluble FcγRIIA. Further mutational analysis identifies the E 253 -R 261 sequence within the α M Idomain as part of the FcγRIIA binding interface within Mac-1. Altogether, these results demonstrate that FcγRIIA recognizes Mac-1 via the α M I-domain but not the lectin domain, a distinct feature from other FcRs, and that Mac-1 binding confers FcγRIIA with the ability to prolong cell adhesion as well as to spread and migrate on the ICs, leading to effective cell killing by ADCC.Receptors recognizing the Fc fragment (FcR) 1 of the immunoglobulin (Ig) molecules are critical to host defense by mediating leukocyte migration toward ICs deposited within the site of infections and by eliciting potent cytolysis of the IC-sensitized targets, such as malignant cells and invading pathogens (1)(2)(3)(4)(5)(6)(7)(8). A number of clinical studies have demonstrated that FcγRIIA (CD32), a major FcR that recognizes the IgG subclass, is essential to the efficacy of several therapeutic antibody-based drugs, including Herceptin for HER-2/neu-positive breast cancer cells, Rituxan for non-hodgkins lymphoma, and the antibodies for melanoma (1,2,9). Yet, inappropriate engagement of FcγRIIA also causes autoimmune diseases in patients undergoing treatment using these therapeutic antibodies (10), and in transgenic mice overexpressing FcγRIIA (11).The FcγRIIA-mediated antibody-dependent cytotoxicity (ADCC) is dependent on both FcγRIIA and the integrin Mac-1 (α M β 2 , CR3, CD11b/CD18) (1,8,12,13). Despite the importance of Mac-1 in the FcγRIIA-mediated leukocyte functions, and the extensive studies conducted on Mac-1 interaction with various FcRs, including FcγRIIIB (CD16), FcαRI (CD89) and FcεRII (CD23), in addition to [14][15][16], the molecular basis underlying Mac-1 recognition of these FcRs is still less understood. Based primarily on inhibition studies using lectin-domain-specific inhibitors such as N-acetyl-D-glucosamine (NADG) (13,15,17), the binding sites within Mac-1 for most of the FcRs are mapped to its lectin-domain in the α subunit. However, as Mac-1's ability to promote FcγRIIA-mediated phagocytosis is insensitive to NADG inhibition (18), the nature of the FcγR...

Alzheimer's disease (AD) is the leading cause of senile dementia, and is a complex disorder. The pathological hallmarks of AD were discovered by Dr. Alois Alzheimer in 1907, and include deposits of amyloid or senile plaques and neurofibrillar tangles. Plaques are composed of a peptide, termed the Abeta peptide, that is derived by proteolytic processing of the amyloid precursor protein (APP), while neurofibrillar tangles result from a hyperphosphorylation of the tau protein. Mechanisms associated with the formation of plaques and neurofibrillar tangles and their respective contributions to the disease process have been intensely investigated. Proteolytic processing of APP that results in the generation of the Abeta peptide is now well understood and is influenced by several proteins. Recent evidence suggests that the Abeta levels are carefully regulated, and several proteases play an important role in removing the Abeta peptide. Finally, it is becoming apparent that several members of the LDL receptor family play important roles in the brain, and may modulate the course of AD.