Antonio Bastone

Synthetic amyloid-β oligomers impair long-term memory independently of cellular prion protein

Inability to form new memories is an early clinical sign of Alzheimer’s disease (AD). There is ample evidence that the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of this disorder. Soluble, bio-derived oligomers of Aβ are proposed as the key mediators of synaptic and cognitive dysfunction, but more tractable models of Aβ−mediated cognitive impairment are needed. Here we report that, in mice, acute intracerebroventricular injections of synthetic Aβ1–42 oligomers impaired consolidation of the long-term recognition memory, whereas mature Aβ1–42 fibrils and freshly dissolved peptide did not. The deficit induced by oligomers was reversible and was prevented by an anti-Aβ antibody. It has been suggested that the cellular prion protein (PrPC) mediates the impairment of synaptic plasticity induced by Aβ. We confirmed that Aβ1–42 oligomers interact with PrPC, with nanomolar affinity. However, PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that Aβ1–42 oligomers are responsible for cognitive impairment in AD and that PrPC is not required.

PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3–/– mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3–/– mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-α-trypsin inhibitor is normal in Ptx3–/– cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor α-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.

Regulation of leukocyte recruitment by the long pentraxin PTX3

Pentraxins are a superfamily of conserved proteins involved in the acute-phase response and innate immunity. Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a key component of the humoral arm of innate immunity that is essential for resistance to certain pathogens. A regulatory role for pentraxins in inflammation has long been recognized, but the underlying mechanisms remain unclear. Here we report that PTX3 bound P-selectin and attenuated neutrophil recruitment at sites of inflammation. PTX3 released from activated leukocytes functioned locally to dampen neutrophil recruitment and regulate inflammation. Antibodies have glycosylation-dependent regulatory effect on inflammation. Therefore, PTX3, which is an essential component of humoral innate immunity, and immunoglobulins share functional outputs, including complement activation, opsonization and, as shown here, glycosylation-dependent regulation of inflammation.

A Recessive Mutation in the APP Gene with Dominant-Negative Effect on Amyloidogenesis

β-Amyloid precursor protein (APP) mutations cause familial Alzheimers disease with nearly complete penetrance. We found an APP mutation [alanine-673→valine-673 (A673V)] that causes disease only in the homozygous state, whereas heterozygous carriers were unaffected, consistent with a recessive Mendelian trait of inheritance. The A673V mutation affected APP processing, resulting in enhanced β-amyloid (Aβ) production and formation of amyloid fibrils in vitro. Co-incubation of mutated and wild-type peptides conferred instability on Aβ aggregates and inhibited amyloidogenesis and neurotoxicity. The highly amyloidogenic effect of the A673V mutation in the homozygous state and its anti-amyloidogenic effect in the heterozygous state account for the autosomal recessive pattern of inheritance and have implications for genetic screening and the potential treatment of Alzheimers disease.

Long Pentraxin PTX3 Upregulates Tissue Factor Expression in Human Endothelial Cells: A Novel Link Between Vascular Inflammation and Clotting Activation

Inflammation is a major contributing factor to atherosclerotic plaque development and ischemic heart disease. PTX3 is a long pentraxin that was recently found to be increased in patients with acute myocardial infarction. Because tissue factor (TF), the in vivo trigger of blood coagulation, plays a dominant role in thrombus formation after plaque rupture, we tested the possibility that PTX3 could modulate TF expression. Human umbilical vein endothelial cells, incubated with endotoxin (lipopolysaccharide) or the inflammatory cytokines interleukin-1&bgr; and tumor necrosis factor-&agr;, expressed TF. The presence of PTX3 increased TF activity and antigen severalfold in a dose-dependent fashion. PTX3 exerted its effect at the transcription level, inasmuch as the increased levels of TF mRNA, mediated by the stimuli, were enhanced in its presence. The increase in mRNA determined by PTX3 originated from an enhanced nuclear binding activity of the transacting factor c-Rel/p65, which was mediated by the agonists and measured by electrophoretic mobility shift assay. The mechanism underlying the increased c-Rel/p65 activity resided in an enhanced degradation of the c-Rel/p65 inhibitory protein I&kgr;B&agr;. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. Our results suggest that PTX3, by increasing TF expression, potentially plays a role in thrombogenesis and ischemic vascular disease.

Synergy between Ficolin-2 and Pentraxin 3 Boosts Innate Immune Recognition and Complement Deposition

The long pentraxin 3 (PTX3) is a multifunctional soluble pattern recognition molecule that is crucial in innate immune protection against opportunistic fungal pathogens such as Aspergillus fumigatus. The mechanisms that mediate downstream effects of PTX3 are largely unknown. However, PTX3 interacts with C1q from the classical pathway of the complement. The ficolins are recognition molecules of the lectin complement pathway sharing structural and functional characteristics with C1q. Thus, we investigated whether the ficolins (Ficolin-1, -2, and -3) interact with PTX3 and whether the complexes are able to modulate complement activation on A. fumigatus. Ficolin-2 could be affinity-isolated from human plasma on immobilized PTX3. In binding studies, Ficolin-1 and particularly Ficolin-2 interacted with PTX3 in a calcium-independent manner. Ficolin-2, but not Ficolin-1 and Ficolin-3, bound A. fumigatus directly, but this binding was enhanced by PTX3 and vice versa. Ficolin-2-dependent complement deposition on the surface of A. fumigatus was enhanced by PTX3. A polymorphism in the FCN2 gene causing a T236M amino acid change in the fibrinogen-like binding domain of Ficolin-2, which affects the binding to GlcNAc, reduced Ficolin-2 binding to PTX3 and A. fumigatus significantly. These results demonstrate that PTX3 and Ficolin-2 may recruit each other on pathogens. The effect was alleviated by a common amino acid change in the fibrinogen-like domain of Ficolin-2. Thus, components of the humoral innate immune system, which activate different complement pathways, cooperate and amplify microbial recognition and effector functions.

Binding of the Long Pentraxin PTX3 to Factor H: Interacting Domains and Function in the Regulation of Complement Activation

The long pentraxin PTX3 is a multifunctional soluble molecule involved in inflammation and innate immunity. As an acute phase protein, PTX3 binds to the classical pathway complement protein C1q, limits tissue damage in inflammatory conditions by regulating apoptotic cell clearance, and plays a role in the phagocytosis of selected pathogens. This study was designed to investigate the interaction of PTX3 with factor H (FH), the main soluble alternative pathway regulatory protein. We report that PTX3 binds FH with an apparent Kd of 1.1 × 10−7 M, and define two binding sites for PTX3 on FH. The primary binding site is located on FH domains 19–20, which interact with the N-terminal domain of PTX3, while a secondary binding site on domain 7 binds the glycosylated PTX3 pentraxin domain. The FH Y402H polymorphism, which affects binding to the short pentraxin CRP, did not affect binding to PTX3. Surface-bound PTX3 enhances FH recruitment and iC3b deposition and PTX3-bound FH retains its activity as a cofactor for factor I-mediated C3b cleavage. Thus, our findings identify PTX3 as a unique FH ligand in that it can bind both of the two hot-spots of FH, namely SCR7 and SCR19–20 and indicate that PTX3 participates in the localization of functionally active FH.

PTX3 Interacts with Inter-α-trypsin Inhibitor IMPLICATIONS FOR HYALURONAN ORGANIZATION AND CUMULUS OOPHORUS EXPANSION

Pentraxin 3 (PTX3) and heavy chains (HCs) of inter-α-trypsin inhibitor (IαI) are essential for hyaluronan (HA) organization within the extracellular matrix of the cumulus oophorus, which is critical for in vivo oocyte fertilization and female fertility. In this study, we examined the possibility that these molecules interact and cooperate in this function. We show that HCs and PTX3 colocalize in the cumulus matrix and coimmunoprecipitate from cumulus matrix extracts. Coimmunoprecipitation experiments and solid-phase binding assays performed with purified human IαI and recombinant PTX3 demonstrate that their interaction is direct and not mediated by other matrix components. PTX3 does not bind to IαI subcomponent bikunin and, accordingly, bikunin does not compete for the binding of PTX3 to IαI, indicating that PTX3 interacts with IαI subcomponent HC only. Recombinant PTX3-specific N-terminal region, but not the PTX3-pentraxin C-terminal domain, showed the same ability as full-length protein to bind to HCs and to enable HA organization and matrix formation by Ptx3-/- cumulus cell oocyte complexes cultured in vitro. Furthermore, a monoclonal antibody raised against PTX3 N terminus, which inhibits PTX3/IαI interaction, also prevents recombinant full-length PTX3 from restoring a normal phenotype to in vitro-cultured Ptx3-/- cumuli. These results indicate that PTX3 directly interacts with HCs of IαI and that such interaction is essential for organizing HA in the viscoelastic matrix of cumulus oophorus, highlighting a direct functional link between the two molecules.

Identification of an Antiangiogenic FGF2-binding Site in the N Terminus of the Soluble Pattern Recognition Receptor PTX3

Long-pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 comprises a pentraxin-like C-terminal domain involved in complement activation via C1q interaction and an N-terminal extension with unknown functions. PTX3 binds fibroblast growth factor-2 (FGF2), inhibiting its pro-angiogenic and pro-restenotic activity. Here, retroviral transduced endothelial cells (ECs) overexpressing the N-terminal fragment PTX3-(1–178) showed reduced mitogenic activity in response to FGF2. Accordingly, purified recombinant PTX3-(1–178) binds FGF2, prevents PTX3/FGF2 interaction, and inhibits FGF2 mitogenic activity in ECs. Also, the monoclonal antibody mAb-MNB4, which recognizes the PTX3-(87–99) epitope, prevents FGF2/PTX3 interaction and abolishes the FGF2 antagonist activity of PTX3. Consistently, the synthetic peptides PTX3-(82–110) and PTX3-(97–110) bind FGF2 and inhibit the interaction of FGF2 with PTX3 immobilized to a BIAcore sensor chip, FGF2-dependent EC proliferation, and angiogenesis in vivo. Thus, the data identify a FGF2-binding domain in the N-terminal extension of PTX3 spanning the PTX3-(97–110) region, pointing to a novel function for the N-terminal extension of PTX3 and underlining the complexity of the PTX3 molecule for modular humoral pattern recognition.

JNK regulates APP cleavage and degradation in a model of Alzheimer's disease

Secretion of Amyloid-beta peptide (Abeta) circulating oligomers and their aggregate forms derived by processing of beta-amyloid precursor protein (APP) are a key event in Alzheimers disease (AD). We show that phosphorylation of APP on threonine 668 may play a role in APP metabolism in H4-APP(sw) cell line, a degenerative AD model. We proved that JNK plays a fundamental role in this phosphorylation since its specific inhibition, with the JNK inhibitor peptide (D-JNKI1), induced APP degradation and prevented APP phosphorylation at T668. This results in a significant drop of betaAPPs, Abeta fragments and Abeta circulating oligomers. Moreover the D-JNKI1 treatment produced a switch in the APP metabolism, since the peptide reduced the rate of the amyloidogenic processing in favour of the non-amyloidogenic one. All together our results suggest an important link between APP metabolism and the JNK pathway and contribute to shed light on the molecular signalling pathway of this disease indicating JNK as an innovative target for AD therapy.