APP fragment controls both ionotropic and non-ionotropic signaling of NMDA receptors.

NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating synaptic long-term depression (LTD) and spine shrinkage. Here, we identify AETA (Aη), an amyloid-ß precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes. AETA inhibits ionotropic NMDAR activity by competing with the co-agonist and induces an intracellular conformational modification of GluN1 subunits. This favors non-ionotropic NMDAR signaling leading to enhanced LTD and favors spine shrinkage. Endogenously, AETA production is increased by in vivo chemogenetically induced neuronal activity. Genetic deletion of AETA production alters NMDAR transmission and prevents LTD, phenotypes rescued by acute exogenous AETA application. This genetic deletion also impairs contextual fear memory. Our findings demonstrate AETA-dependent NMDAR activation (ADNA), characterizing AETA as a unique type of endogenous NMDAR modulator that exerts bidirectional control over NMDAR signaling and associated information processing.

Beneficial Effect of ACI-24 Vaccination on Aß Plaque Pathology and Microglial Phenotypes in an Amyloidosis Mouse Model.

Amyloid-ß (Aß) deposition is an initiating factor in Alzheimer's disease (AD). Microglia are the brain immune cells that surround and phagocytose Aß plaques, but their phagocytic capacity declines in AD. This is in agreement with studies that associate AD risk loci with genes regulating the phagocytic function of immune cells. Immunotherapies are currently pursued as strategies against AD and there are increased efforts to understand the role of the immune system in ameliorating AD pathology. Here, we evaluated the effect of the Aß targeting ACI-24 vaccine in reducing AD pathology in an amyloidosis mouse model. ACI-24 vaccination elicited a robust and sustained antibody response in APPPS1 mice with an accompanying reduction of Aß plaque load, Aß plaque-associated ApoE and dystrophic neurites as compared to non-vaccinated controls. Furthermore, an increased number of NLRP3-positive plaque-associated microglia was observed following ACI-24 vaccination. In contrast to this local microglial activation at Aß plaques, we observed a more ramified morphology of Aß plaque-distant microglia compared to non-vaccinated controls. Accordingly, bulk transcriptomic analysis revealed a trend towards the reduced expression of several disease-associated microglia (DAM) signatures that is in line with the reduced Aß plaque load triggered by ACI-24 vaccination. Our study demonstrates that administration of the Aß targeting vaccine ACI-24 reduces AD pathology, suggesting its use as a safe and cost-effective AD therapeutic intervention.

Aη-α and Aη-ß peptides impair LTP ex vivo within the low nanomolar range and impact neuronal activity in vivo.

BACKGROUND: Amyloid precursor protein (APP) processing is central to Alzheimer's disease (AD) etiology. As early cognitive alterations in AD are strongly correlated to abnormal information processing due to increasing synaptic impairment, it is crucial to characterize how peptides generated through APP cleavage modulate synapse function. We previously described a novel APP processing pathway producing η-secretase-derived peptides (Aη) and revealed that Aη-α, the longest form of Aη produced by η-secretase and α-secretase cleavage, impaired hippocampal long-term potentiation (LTP) ex vivo and neuronal activity in vivo. METHODS: With the intention of going beyond this initial observation, we performed a comprehensive analysis to further characterize the effects of both Aη-α and the shorter Aη-ß peptide on hippocampus function using ex vivo field electrophysiology, in vivo multiphoton calcium imaging, and in vivo electrophysiology. RESULTS: We demonstrate that both synthetic peptides acutely impair LTP at low nanomolar concentrations ex vivo and reveal the N-terminus to be a primary site of activity. We further show that Aη-ß, like Aη-α, inhibits neuronal activity in vivo and provide confirmation of LTP impairment by Aη-α in vivo. CONCLUSIONS: These results provide novel insights into the functional role of the recently discovered η-secretase-derived products and suggest that Aη peptides represent important, pathophysiologically relevant, modulators of hippocampal network activity, with profound implications for APP-targeting therapeutic strategies in AD.

Transgenic Overexpression of the Disordered Prion Protein N1 Fragment in Mice Does Not Protect Against Neurodegenerative Diseases Due to Impaired ER Translocation.

The structurally disordered N-terminal half of the prion protein (PrPC) is constitutively released into the extracellular space by an endogenous proteolytic cleavage event. Once liberated, this N1 fragment acts neuroprotective in ischemic conditions and interferes with toxic peptides associated with neurodegenerative diseases, such as amyloid-beta (Aß) in Alzheimer's disease. Since analog protective effects of N1 in prion diseases, such as Creutzfeldt-Jakob disease, have not been studied, and given that the protease releasing N1 has not been identified to date, we have generated and characterized transgenic mice overexpressing N1 (TgN1). Upon intracerebral inoculation of TgN1 mice with prions, no protective effects were observed at the levels of survival, clinical course, neuropathological, or molecular assessment. Likewise, primary neurons of these mice did not show protection against Aß toxicity. Our biochemical and morphological analyses revealed that this lack of protective effects is seemingly due to an impaired ER translocation of the disordered N1 resulting in its cytosolic retention with an uncleaved signal peptide. Thus, TgN1 mice represent the first animal model to prove the inefficient ER translocation of intrinsically disordered domains (IDD). In contrast to earlier studies, our data challenge roles of cytoplasmic N1 as a cell penetrating peptide or as a potent "anti-prion" agent. Lastly, our study highlights both the importance of structured domains in the nascent chain for proteins to be translocated and aspects to be considered when devising novel N1-based therapeutic approaches against neurodegenerative diseases.

Identification of a rare presenilin 1 single amino acid deletion mutation (F175del) with unusual amyloid-ß processing effects.

We report the novel presenilin 1 (PSEN1) single amino acid deletion mutation F175del. Comprehensive clinical work-up, including cerebral MRI, FDG-PET, and CSF analysis, was performed in a male who had developed forgetfulness at the age of 39. Alzheimer's disease dementia was diagnosed according to established criteria. The index patient manifested rapid progressive dementia, seizures, and myoclonus, and a Pisa syndrome as a side effect of donepezil treatment. The PSEN1 mutation F175del was found on genetic testing. It was rendered very likely pathogenic as amyloid-ß (Aß) peptide 42 was elevated in a cell culture model compared to presenilin 1 wild-type controls. An additional, unusual increase in Aß39 indicates a rarely observed product line deviation in the generation of the shorter Aß species. Our observations extend the range of PSEN1 mutations to be considered in familial dementia. We demonstrate that deletion of a single conserved amino acid, which is very rare compared to missense mutations as the common cause for PSEN1-associated Alzheimer's disease, can lead to an unusual profile of Aß species.

Aß-induced acceleration of Alzheimer-related τ-pathology spreading and its association with prion protein.

Extracellular deposition of amyloid ß-protein (Aß) in amyloid plaques and intracellular accumulation of abnormally phosphorylated τ-protein (p-τ) in neurofibrillary tangles (NFTs) represent pathological hallmark lesions of Alzheimer's disease (AD). Both lesions develop in parallel in the human brain throughout the preclinical and clinical course of AD. Nevertheless, it is not yet clear whether there is a direct link between Aß and τ pathology or whether other proteins are involved in this process. To address this question, we crossed amyloid precursor protein (APP) transgenic mice overexpressing human APP with the Swedish mutation (670/671 KM → NL) (APP23), human wild-type APP (APP51/16), or a proenkephalin signal peptide linked to human Aß42 (APP48) with τ-transgenic mice overexpressing human mutant 4-repeat τ-protein with the P301S mutation (TAU58). In 6-month-old APP23xTAU58 and APP51/16xTAU58 mice, soluble Aß was associated with the aggravation of p-τ pathology propagation into the CA1/subiculum region, whereas 6-month-old TAU58 and APP48xTAU58 mice neither exhibited significant amounts of p-τ pathology in the CA1/subiculum region nor displayed significant levels of soluble Aß in the forebrain. In APP23xTAU58 and APP51/16xTAU58 mice showing an acceleration of p-τ propagation, Aß and p-τ were co-immunoprecipitated with cellular prion protein (PrPC). A similar interaction between PrPC, p-τ and Aß was observed in human AD brains. This association was particularly noticed in 60% of the symptomatic AD cases in our sample, suggesting that PrPC may play a role in the progression of AD pathology. An in vitro pull-down assay confirmed that PrPC is capable of interacting with Aß and p-τ. Using a proximity ligation assay, we could demonstrate proximity (less than ~ 30-40 nm distance) between PrPC and Aß and between PrPC and p-τ in APP23xTAU58 mouse brain as well as in human AD brain. Proximity between PrPC and p-τ was also seen in APP51/16xTAU58, APP48xTAU58, and TAU58 mice. Based on these findings, it is tempting to speculate that PrPC is a critical player in the interplay between Aß and p-τ propagation at least in a large group of AD cases. Preexisting p-τ pathology interacting with PrPC, thereby, appears to be a prerequisite for Aß to function as a p-τ pathology accelerator via PrPC.

Physiological and pathophysiological control of synaptic GluN2B-NMDA receptors by the C-terminal domain of amyloid precursor protein.

The amyloid precursor protein (APP) harbors physiological roles at synapses and is central to Alzheimer's disease (AD) pathogenesis. Evidence suggests that APP intracellular domain (AICD) could regulate synapse function, but the underlying molecular mechanisms remain unknown. We addressed AICD actions at synapses, per se, combining in vivo AICD expression, ex vivo AICD delivery or APP knock-down by in utero electroporation of shRNAs with whole-cell electrophysiology. We report a critical physiological role of AICD in controlling GluN2B-containing NMDA receptors (NMDARs) at immature excitatory synapses, via a transcription-dependent mechanism. We further show that AICD increase in mature neurons, as reported in AD, alters synaptic NMDAR composition to an immature-like GluN2B-rich profile. This disrupts synaptic signal integration, via over-activation of SK channels, and synapse plasticity, phenotypes rescued by GluN2B antagonism. We provide a new physiological role for AICD, which becomes pathological upon AICD increase in mature neurons. Thus, AICD could contribute to AD synaptic failure.

Proteolytic Processing of Neuregulin 1 Type III by Three Intramembrane-cleaving Proteases.

Numerous membrane-bound proteins undergo regulated intramembrane proteolysis. Regulated intramembrane proteolysis is initiated by shedding, and the remaining stubs are further processed by intramembrane-cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of ß-secretase (ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1)). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Because of the hairpin nature of NRG1 type III, two membrane-bound stubs with a type 1 and a type 2 orientation are generated by proteolytic processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1-oriented stub is further cleaved by γ-secretase at an ϵ-like site five amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ϵ-cleavage site is only one amino acid N-terminal to a Val/Leu substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III ß-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer disease-associated mutations within the amyloid precursor protein. The type 2-oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with 2,2'-(2-oxo-1,3-propanediyl)bis[(phenylmethoxy)carbonyl]-l-leucyl-l-leucinamide ketone inhibits formation of N-terminal intracellular domains and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate that is not only cleaved by multiple sheddases but is also processed by three different I-CLiPs.

η-Secretase processing of APP inhibits neuronal activity in the hippocampus.

Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid-ß peptide. Two principal physiological pathways either prevent or promote amyloid-ß generation from its precursor, ß-amyloid precursor protein (APP), in a competitive manner. Although APP processing has been studied in great detail, unknown proteolytic events seem to hinder stoichiometric analyses of APP metabolism in vivo. Here we describe a new physiological APP processing pathway, which generates proteolytic fragments capable of inhibiting neuronal activity within the hippocampus. We identify higher molecular mass carboxy-terminal fragments (CTFs) of APP, termed CTF-η, in addition to the long-known CTF-α and CTF-ß fragments generated by the α- and ß-secretases ADAM10 (a disintegrin and metalloproteinase 10) and BACE1 (ß-site APP cleaving enzyme 1), respectively. CTF-η generation is mediated in part by membrane-bound matrix metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504-505 of APP695, releasing a truncated ectodomain. After shedding of this ectodomain, CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (termed Aη-α and Aη-ß). CTFs produced by η-secretase are enriched in dystrophic neurites in an AD mouse model and in human AD brains. Genetic and pharmacological inhibition of BACE1 activity results in robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor, hippocampal long-term potentiation was reduced. Notably, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo, long-term potentiation was lowered. Furthermore, in vivo single-cell two-photon calcium imaging showed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major functionally relevant APP processing pathway, but may also indicate potential translational relevance for therapeutic strategies targeting APP processing.

Laminin-121--recombinant expression and interactions with integrins.

Laminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, ß2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm-Swarm tumor (EHS-laminin) but its T(m) value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the ß chain reduced the stability of the coiled-coil structure of laminin-121. Its binding to integrins was compared with EHS-laminin, laminin-3A32 purified from murine epidermal cell line and recombinantly expressed laminins-111, -211 and -221. Laminin-121 showed the highest affinity to α6ß1 and α7ß1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the ß2 laminins have higher affinity for integrins than the ß1 laminins.

Structural basis of sequence-specific collagen recognition by SPARC.

Protein interactions with the collagen triple helix play a critical role in collagen fibril formation, cell adhesion, and signaling. However, structural insight into sequence-specific collagen recognition is limited to an integrin-peptide complex. A GVMGFO motif in fibrillar collagens (O denotes 4-hydroxyproline) binds 3 unrelated proteins: von Willebrand factor (VWF), discoidin domain receptor 2 (DDR2), and the extracellular matrix protein SPARC/osteonectin/BM-40. We report the crystal structure at 3.2 A resolution of human SPARC bound to a triple-helical 33-residue peptide harboring the promiscuous GVMGFO motif. SPARC recognizes the GVMGFO motifs of the middle and trailing collagen chains, burying a total of 720 A(2) of solvent-accessible collagen surface. SPARC binding does not distort the canonical triple helix of the collagen peptide. In contrast, a critical loop in SPARC is substantially remodelled upon collagen binding, creating a deep pocket that accommodates the phenylalanine residue of the trailing collagen chain ("Phe pocket"). This highly restrictive specificity pocket is shared with the collagen-binding integrin I-domains but differs strikingly from the shallow collagen-binding grooves of the platelet receptor glycoprotein VI and microbial adhesins. We speculate that binding of the GVMGFO motif to VWF and DDR2 also results in structural changes and the formation of a Phe pocket.

Mapping of SPARC/BM-40/osteonectin-binding sites on fibrillar collagens.

The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix alphaA of the extracellular calcium-binding domain of SPARC and is partially masked by helix alphaC. Previously, we found that the removal of helix alphaC caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site approximately 180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven alpha2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOGPSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among alpha chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo.

Fibromodulin interactions with type I and II collagens.

Fibromodulin is a keratan-sulfate small leucine-rich proteoglycan (SLRP) regulating collagen I and II fibril formation. In vivo studies suggest that, alongside decorin, fibromodulin plays an important role in the maintenance of mature tissues. To characterize fibromodulin/decorin differences in binding to type I and II collagen, we tested the collagen CNBr peptides in solid-phase assays. Only one peptide from collagen II and several peptides from collagen I interacted with fibromodulin, pointing to multiple binding sites in the collagen I molecule. By Scatchard-type analysis, the fibromodulin molecule showed only one class of binding sites for collagen I and both low and high affinity (classes of) binding sites for collagen II. Lys/Hyl residues in both collagens are essential for the interaction. Fibril formation assays showed the concomitant presence of fibromodulin and decorin in fibrils and a cumulative inhibitory effect. In solid-phase assays decorin seems to inhibit fibromodulin binding, whereas the contrary does not occur. We found fibromodulin and decorin have similarities and differences that may represent the biochemical basis of redundancy in SLRP function with compensation between different (classes of) SLRPs.

Molecular stability of chemically modified collagen triple helices.

Ionic residues influence the stability of collagen triple helices and play a relevant role in the spontaneous aggregation of fibrillar collagens. Collagen types I and II and some of their CNBr peptides were chemically modified in mild conditions with two different protocols. Primary amino groups of Lys and Hyl were N-methylated by formaldehyde in reducing conditions or N-acetylated by sulfosuccinimidyl acetate. The positive charge of amino groups at physiological pH was maintained after the former modification, whereas it was lost after the latter. These chemical derivatizations did not significantly alter the stability of the triple helical conformation of peptide trimeric species. Also the enthalpic change on denaturation was largely unaffected by derivatizations. This implies that no significant variation of weak bonds, either in number or overall strength, and of entropy occur on modification. These properties can probably be explained by the fact that chemically modified groups maintain the ability to form hydrogen bonds.

Platelet interaction with CNBr peptides from type II collagen via integrin alpha2beta1.

Adhesion of blood platelets to fibrillar collagens plays a crucial role in haemostasis. Collagen type II is a homotrimeric member of the fibrillar collagen family, whose ability to interact with platelets has been poorly investigated. In this work, we analysed platelet adhesion to the whole collagen type II molecule, as well as to its CNBr peptides. We found that collagen type II is as efficient as collagen type I in supporting platelet adhesion. Platelet binding sites on collagen type II were identified in two different CNBr-derived peptides, CB8 and CB11. The ability of these peptides to support platelet adhesion required the triple helical conformation. Interaction of platelets with CB8 and CB11 peptides was totally dependent on the presence of Mg(2+) ions, and was completely inhibited by the anti-integrin alpha(2)beta(1) antibody P1E6. Upon adhesion to CB8 and CB11, a significant increase in intracellular protein tyrosine phosphorylation was observed. The pattern of tyrosine phosphorylated proteins in CB8- and CB11-adherent platelets was very similar to that observed in platelets adherent to the whole collagen molecule. By immunoprecipitation experiments, we identified two substrates that were tyrosine phosphorylated in adherent platelets as the tyrosine kinase Syk and the PLCgamma2 isozyme. By contrast, platelet adhesion to CB8 and CB11 did not promote tyrosine phosphorylation of FcR gamma-chain. Finally, we found that collagen type II, but not the CNBr-derived peptides, was able to induce cell aggregation associated to protein tyrosine phosphorylation when added to a platelet suspension. These results identify the CNBr peptides from collagen type II CB8 and CB11 as ligands for platelet integrin alpha(2)beta(1), and recognise their ability to support platelet adhesion and activation.

Interaction of decorin with CNBr peptides from collagens I and II. Evidence for multiple binding sites and essential lysyl residues in collagen.

Decorin is a small leucine-rich chondroitin/dermatan sulfate proteoglycan reported to interact with fibrillar collagens through its protein core and to localize at d and e bands of the collagen fibril banding pattern. Using a solid-phase assay, we have determined the interaction of peptides derived by CNBr cleavage of type I and type II collagen with decorin extracted from bovine tendon and its protein core and with a recombinant decorin preparation. At least five peptides have been found to interact with all three decorin samples. The interaction of peptides with tendon decorin has a dissociation constant in the nanomolar range. The triple helical conformation of the peptide trimeric species is a necessary requisite for the binding. All positive peptides have a region within the d and e bands of collagen fibrils. Two chemical derivatives of collagens and of positive peptides were prepared by N-acetylation and N-methylation of the primary amino group of Lys/Hyl side chains. Chemical modifications performed in mild conditions do not significantly alter the thermal stability of peptide trimeric species whereas they affect the interaction with decorin: N-acetylation eliminates both the positive charge and the binding to decorin, whereas N-methylation preserves the cationic character and modulates the binding. We conclude that decorin makes contacts with multiple sites in type I collagen and probably also in type II collagen and that some collagen Lys/Hyl residues are essential for the binding.