Domain Of Amyloid Precursor Protein Research Articles

Nanodisc Reconstitution and Characterization of Amyloid-β Precursor Protein C99.

Amyloid precursor protein (APP) plays a pivotal role in the pathology of Alzheimer's disease (AD). Since the fragmentation of the membrane-bound APP that results in the production of amyloid-β peptides is the starting point for amyloid toxicity in AD, it is important to investigate the structure and dynamics of APP in a near-native lipid-bilayer environment. However, the reconstitution of APP into a stable and suitable membrane-mimicking lipid environment is a challenging task. In this study, the 99-residue C-terminal domain of APP is successfully reconstituted into polymer nanodiscs and characterized using size-exclusion chromatography, mass spectrometry, solution NMR, and magic-angle spinning solid-state NMR. In addition, the feasibility of using lipid-solubilizing polymers for isolating and characterizing APP in the native Escherichia. coli membrane environment is demonstrated.

Ethnogenetic-specific mutations in Alzheimer’s disease: A marker of clinical outcomes

Alzheimer’s disease (AD) presents a substantial global health challenge, with its pathogenesis influenced by a complex interplay of genetic and molecular factors. Approximately 1% of AD cases are attributed to early onset autosomal dominant familial AD (fAD), with genetics contributing to about 70% of overall AD risk. Understanding the genetic basis and molecular mechanisms is paramount for early diagnosis and prognosis improvement. This study explores a previously underexplored area of fAD, examining how different mutations within the same gene, encoding a single protein, may influence the clinical features and progression of fAD subtypes. Our investigation focuses on distinct fAD subtypes — Iranian (T714A), Swedish (KM670/671NL), and Australian (L723P) — and their associated mutations within the C-terminus domain of amyloid precursor protein (APP). Through an extensive analysis of existing literature encompassing clinical severity, pathogenicity, neuropathology, and biological processes, we reveal critical insights into these fAD subtypes. Leveraging bioinformatics tools, we correlate the physicochemical properties of translated mutant proteins with clinical and neuropathological features. Notably, our findings demonstrate that mutations occurring between codons 714 and 717 of the APP gene share a higher similarity, resulting in lower root mean squared deviation scores. These mutations are associated with a broader spectrum of clinical symptoms, including myoclonus and seizures, and an earlier age of onset. Moreover, we observe a direct correlation between the location of genetic mutations on the protein sequence and specific physicochemical properties, clinical presentations, and neuropathological features among fAD subtypes. Mutations with higher structural similarity tend to manifest similar clinical and physical characteristics. While certain neuropathological findings correlate with an increasing epitope toxicity burden, our analysis indicates that epitope toxicity does not significantly impact clinical outcomes. In summary, our study provides novel insights into the heterogeneous nature of fAD subtypes, illuminating the intricate relationship between genetic mutations, physicochemical properties, and clinical manifestations. These findings offer a foundation for further research into tailored therapeutic approaches and personalized medicine for fAD.

Co-treatment with the seed of Carthamus tinctorius L. and the aerial part of Taraxacum coreanum synergistically suppresses Aβ25-35-induced neurotoxicity by altering APP processing.

Accumulation of β-amyloid peptide (Aβ) induces neurotoxicity, which is the primary risk factor in the pathogenesis of Alzheimer's disease (AD). The cleavage of amyloid precursor protein (APP) by the β- (BACE) and γ- (PS1, PS2) secretases is a critical step in the amyloidogenic pathway. The induction of neuronal apoptosis by Aβ involves increased expression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and decreased Bcl-2 expression. The seed of Carthamus tinctorius L. (CTS) and the aerial part of Taraxacum coreanum (TC) are traditional herbs used to treat several neurodegenerative diseases. In this study, the neuroprotective effects of co-treatment with CTS and TC on Aβ-induced neurotoxicity in SH-SY5Y neuroblastoma cells and the underlying mechanisms were investigated. CTS, TC, and the co-treatment (CTS + TC) were added to Aβ25-35-treated SH-SY5Y cells. CTS + TC synergistically increased cell viability and inhibited reactive oxygen species production. CTS + TC resulted in significant downregulation of BACE, PS1, PS2, and APP, as well as the 99-aa C-terminal domain of APP, compared with either CTS or TC alone. Compared with the single herbs, co-treatment with CTS and TC markedly decreased the expression of Bax and increased the expression of Bcl-2, consistent with its anti-apoptotic effects. These findings suggest that co-treatment with CTS and TC may be useful for AD prevention.

Revisit the E2 Domain of Amyloid Precursor Protein: Ferroxidase, Superoxide and Peroxynitrite Scavenging Activities.

Amyloid precursor protein (APP) is the biological precursor of β-amyloids, a known histopathological hallmark associated with Alzheimer's disease (AD). The function of APP is of great interest yet remains elusive. One of the extracellular domains of APP, the E2 domain, has been proposed to possess ferroxidase activity and affect neuronal iron homeostasis. However, contradicting evidence has been reported, and its precise role remains inconclusive. Here, we studied the Cu-binding site of the E2 domain using extended X-ray absorption fine structure (EXAFS), UV-vis, and electron paramagnetic resonance (EPR) and discovered that a new labile water ligand coordinates to the Cu(II) cofactor in addition to the four known histidines. We explored the proposed ferroxidase activity of the Cu(II)-E2 domain through reactions with ferrous iron and observed single-turnover ferrous oxidation activity with a rate up to 1.0 × 102 M-1 s-1. Cu(I)-E2 reacted with molecular oxygen at a rate of only 5.3 M-1 s-1, which would restrict any potential multiturnover ferroxidase activity to this slow rate and prevents observation of activity under multiturnover conditions. The positive electrostatic potential surface of the protein indicates possible reactivity with negatively charged small substrates such as superoxide radicals (O2•-) and peroxynitrite (ONOO-) that are major contributors to the oxidative stress prevalent in the extracellular environment. Our assays showed that Cu(I)-E2 can remove O2•- at a rate of 1.6 × 105 M-1 s-1, which is slower than the rates of native SODs. However, the reaction between Cu(I)-E2 and ONOO- achieved a rate of 1.1 × 105 M-1 s-1, comparable to native ONOO- scavenger peroxiredoxins (105-107 M-1 s-1). Therefore, the E2 domain of APP can serve as an enzymatic site that may function as a ferroxidase under substrate-limiting conditions, a supplemental O2•- scavenger, and an ONOO- remover in the vicinity of the cellular iron efflux channel and protect neuron cells from reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage.

Amyloid beta accumulations and enhanced neuronal differentiation in cerebral organoids of Dutch-type cerebral amyloid angiopathy patients.

ADutch-type cerebral amyloid angiopathy (D-CAA) is a hereditary brain disorder caused by a point mutation in the amyloid precursor protein (APP) gene. The mutation is located within the amyloid beta (Aβ) domain of APP and leads to Aβ peptide accumulation in and around the cerebral vasculature. There lack of disease models to study the cellular and molecular pathological mechanisms of D-CAA together with the absence of a disease phenotype in vitro in overexpression cell models, as well as the limited availability of D-CAA animal models indicates the need for a D-CAA patient-derived model. We generated cerebral organoids from four D-CAA patients and four controls, cultured them up to 110 days and performed immunofluorescent and targeted gene expression analyses at two time points (D52 and D110). D-CAA cerebral organoids exhibited Aβ accumulations, showed enhanced neuronal and astrocytic gene expression and TGFβ pathway de-regulation. These results illustrate the potential of cerebral organoids as in vitro disease model of D-CAA that can be used to understand disease mechanisms of D-CAA and can serve as therapeutic intervention platform for various Aβ-related disorders.

Formation of extramembrane β-strands controls dimerization of transmembrane helices in amyloid precursor protein C99

The 99-residue C-terminal domain of amyloid precursor protein (APP-C99), precursor to amyloid beta (Aβ), is a transmembrane (TM) protein containing intrinsically disordered N- and C-terminal extramembrane domains. Using molecular dynamics (MD) simulations, we show that the structural ensemble of the C99 monomer is best described in terms of thousands of states. The C99 monomer has a propensity to form β-strand in the C-terminal extramembrane domain, which explains the slow spin relaxation times observed in paramagnetic probe NMR experiments. Surprisingly, homodimerization of C99 not only narrows the conformational ensemble from thousands to a few states through the formation of metastable β-strands in extramembrane domains but also stabilizes extramembrane α-helices. The extramembrane domain structure is observed to dramatically impact the homodimerization motif, resulting in the modification of TM domain conformations. Our study provides an atomic-level structural basis for communication between the extramembrane domains of the C99 protein and TM homodimer formation. This finding could serve as a general model for understanding the influence of disordered extramembrane domains on TM protein structure.

Covalent fragment inhibits intramembrane proteolysis.

Alzheimer’s disease (AD) is a serious public health crisis with only one current modifying treatment. The reduction of amyloid load by targeting γ-secretase (GS) has been a leading approach in AD drug discovery and development. Despite the focus on GS inhibition, multiple GS inhibitors (GSIs) have failed in clinical trials as a result of side effects including exacerbated cognitive decline. These side effects are largely attributable to inhibition of normal GS function. Standard enzyme inhibitors target catalytic or allosteric sites of the enzyme, including the active site presenilin, as previous GSIs did. To avoid issues observed from broad-spectrum GSIs we discovered that fragment 6H8 that covalently binds to the substrate of GS, the transmembrane domain of amyloid precursor protein (APPTM). Nuclear Magnetic Resonance (NMR) Spectroscopy combined with MALDI-TOF-MS established 6H8 covalently binds to APPTM. 6H8 acts as a Michael acceptor and covalently links to the side chain amines of lysine residues, specifically targeting a cluster of C-terminal lysines K53–K55. Through this modification, 6H8 can inhibit intramembrane proteolysis of an archaeal homolog of presenilin (the active subunit of GS) via substrate binding with a 2–4 μM IC50, determined by a gel-based cleavage assay. 6H8, while too small to be an effective drug candidate, can be combined with a specific non-covalent partner and function as an effective covalent warhead of a targeted covalent inhibitor (TCI). The future development of the 6H8 fragment into the covalent warhead of a TCI is, to our knowledge, a novel approach to AD drug discovery.

Live Cell Fluorescence Imaging Shows Neurotransmitter Activation Promotes Aggregation of the Intracellular Domain of Amyloid Precursor Protein.

Amyloid precursor protein (APP) is a major contributor to the pathology of Alzheimer's and other neurodegenerative diseases through the accumulation of extracellular plaques. Here, we have studied changes in APP translation and aggregation of the APP intracellular domain when the Gαq/PLCβ signaling system is activated by neurotransmitters. Using RT-PCR and a molecular beacon that follows APP mRNA in live cells, we find that Gαq activation sequesters APP mRNA similar to the stress granule response found in heat shock and hypo-osmotic shock thereby shutting down the production of APP. Following the intracellular domain of eGFP-APP, we find that Gαq stimulation increases aggregation as followed by number and brightness (N&B) analysis of single molecule fluorescence time series. Additionally, we show that APP aggregation is affected by changes in the levels of PLCβ1 and its cytosolic binding partners. Our studies show the neurotransmitter activation of Gαq/PLCβ reduces translation of APP and increases aggregation of its intracellular domain. These studies better establish a link between APP production and complexation and Gαq stimulation.

The transmembrane domain of the amyloid precursor protein is required for antiamyloidogenic processing by α-secretase ADAM10

Neurotoxic amyloid β-peptides are thought to be a causative agent of Alzheimer’s disease in humans. The production of amyloid β-peptides from amyloid precursor protein (APP) could be diminished by enhancing α-processing; however, the physical interactions between APP and α-secretases are not well understood. In this study, we employed super-resolution light microscopy to examine in cell-free plasma membranes the abundance and association of APP and α-secretases ADAM10 (a disintegrin and metalloproteinase) and ADAM17. We found that both secretase molecules localize similarly closely to APP (within ≤50 nm). However, when cross-linking APP with antibodies directed against the GFP tag of APP, in confocal microscopy, we observed that only ADAM10 coaggregated with APP. Furthermore, we mapped the involved protein domain by using APP variants with an exchanged transmembrane segment or lacking cytoplasmic/extracellular domains. We identified that the transmembrane domain of APP is required for association with α-secretases and, as analyzed by Western blot, for α-processing. We propose that the transmembrane domain of APP interacts either directly or indirectly with ADAM10, but not with ADAM17, explaining the dominant role of ADAM10 in α-processing of APP. Further understanding of this interaction may facilitate the development of a therapeutic strategy based on promoting APP cleavage by α-secretases.

Mechanism of Tripeptide Trimming of Amyloid β-Peptide 49 by γ-Secretase.

The membrane-embedded γ-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid β-peptides (Aβ) of Alzheimer's disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by γ-secretase remains poorly understood. Here, mass spectrometry and Western blotting were used to quantify the efficiency of tripeptide trimming of wild-type (WT) and familial AD (FAD) mutant Aβ49. In comparison to WT Aβ49, the efficiency of tripeptide trimming was similar for the I45F, A42T, and V46F Aβ49 FAD mutants but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate the tripeptide trimming of Aβ49 by γ-secretase. The starting structure was the active γ-secretase bound to Aβ49 and APP intracellular domain (AICD), as generated from our previous study that captured the activation of γ-secretase for the initial endoproteolytic cleavage of APP (Bhattarai, A., ACS Cent. Sci. 2020, 6, 969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of Aβ49 to Aβ46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and Aβ49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming of Aβ49 by γ-secretase.

Mechanism of tripeptide trimming by γ-secretase

The membrane-embedded γ-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid β-peptides (Aβ) of Alzheimer's disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by γ-secretase remains poorly understood. Here, mass spectrometry and western blotting were used to quantify the efficiency of the first tripeptide trimming step (Aβ49→Aβ46) of wildtype (WT) and familial AD (FAD) mutant APP substrate.

MDGA1 negatively regulates amyloid precursor protein–mediated synapse inhibition in the hippocampus

Balanced synaptic inhibition, controlled by multiple synaptic adhesion proteins, is critical for proper brain function. MDGA1 (meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu [MAM] domain-containing glycosylphosphatidylinositol anchor protein 1) suppresses synaptic inhibition in mammalian neurons, yet the molecular mechanisms underlying MDGA1-mediated negative regulation of GABAergic synapses remain unresolved. Here, we show that the MDGA1 MAM domain directly interacts with the extension domain of amyloid precursor protein (APP). Strikingly, MDGA1-mediated synaptic disinhibition requires the MDGA1 MAM domain and is prominent at distal dendrites of hippocampal CA1 pyramidal neurons. Down-regulation of APP in presynaptic GABAergic interneurons specifically suppressed GABAergic, but not glutamatergic, synaptic transmission strength and inputs onto both the somatic and dendritic compartments of hippocampal CA1 pyramidal neurons. Moreover, APP deletion manifested differential effects in somatostatin- and parvalbumin-positive interneurons in the hippocampal CA1, resulting in distinct alterations in inhibitory synapse numbers, transmission, and excitability. The infusion of MDGA1 MAM protein mimicked postsynaptic MDGA1 gain-of-function phenotypes that involve the presence of presynaptic APP. The overexpression of MDGA1 wild type or MAM, but not MAM-deleted MDGA1, in the hippocampal CA1 impaired novel object-recognition memory in mice. Thus, our results establish unique roles of APP-MDGA1 complexes in hippocampal neural circuits, providing unprecedented insight into trans-synaptic mechanisms underlying differential tuning of neuronal compartment-specific synaptic inhibition.

Conformational Models of APP Processing by Gamma Secretase Based on Analysis of Pathogenic Mutations.

Proteolytic processing of amyloid precursor protein (APP) plays a critical role in the pathogenesis of Alzheimer’s disease (AD). Sequential cleavage of APP by β and γ secretases leads to the generation of Aβ40 (non-amyloidogenic) and Aβ42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) play the role of a catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in an increased Aβ42:Aβ40 ratio and the accumulation of toxic Aβ42 oligomers and plaques in patient brains. In this study, we perform molecular modeling of the APP complex with γ-secretase and analyze potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in the APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures, we propose that APP can form a complex with γ-secretase in 2 potential conformations—M1 and M2. In conformation, the M1 transmembrane domain of APP forms a contact with the perimembrane domain that follows transmembrane domain 6 (TM6) in the PS1 structure. In conformation, the M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in the PS1 structure. By analyzing the effects of PS1-FAD mutations on the local protein disorder index, we discovered that these mutations increase the conformational flexibility of M2 and reduce the conformational flexibility of M1. Based on these results, we propose that M2 conformation, but not M1 conformation, of the γ secretase complex with APP leads to the amyloidogenic (Aβ42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by curved membranes, such as the membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by relatively flat membranes, such as membranes of late endosomes and plasma membranes. These predictions are consistent with published biochemical analyses of APP processing at different subcellular locations. Our results also suggest that specific inhibitors of Aβ42 production could be potentially developed by selectively targeting the M2 conformation of the γ secretase complex with APP.

Abstract 492: CNTN4 is a novel immune checkpoint protein that inhibits proliferation of T cell by interacting with APP

Abstract Contactin 4 (CNTN4, alias BIG-2) is a glycosylphosphatidylinositol (GPI)-anchored neuronal membrane protein that functions as cell adhesion, playing a key role in maintaining the mechanical integrity and signaling properties of the synapse. However, its function and crosstalk with immune cells have not been investigated. Therefore, we tried to find out what role CNTN4 plays in cell adhesion between T cells and antigen-presenting cells (APC). CNTN4 has several binding partners such as the amyloid precursor protein (APP), amyloid-like protein 1 (APLP1), and protein tyrosine phosphatase receptor γ (PTPRγ). In previous papers, the APP was reported to be expressed in T cells. We investigated whether CNTN4 can bind to T cells. The degree of binding of CNTN4 to T cells varied depending on the expression level of APP, and that the binding between these two proteins was in the form of trans-binding. And it was confirmed that the direct binding is achieved through fibronectin 1~2 domain of CNTN4 and KPI (Kunitz protease inhibitor) domain of APP. Furthermore, we investigated whether CNTN4 has any effect on T cell proliferation. T cell proliferation and cytokine secretion assays by using CNTN4 recombinant protein showed that CNTN4 significantly inhibits proliferation and cytokine secretion (IL-2, IFN-γ, TNF-α) of T cells more than PD-L1. Although the expression of CNTN4 in immune cells has not yet been clearly confirmed, the expression of CNTN4 in various carcinomas have confirmed by using western, IHC, and bioinformatic data analysis. In particular, the higher the expression of CNTN4 in tumor tissues of gastric cancer patients, the worse the prognosis is in the survival curve. Our findings suggest that CNTN4 may behave as an immune checkpoint protein similar to other B7 family members. Further investigation of the CNTN4 signaling pathway and its receptors are warranted by our data and may lead to novel therapeutic interventions. Citation Format: Mi Young Cha, Bu-Nam Jeon, Areum Jeong, Joo-Yeon Chung, Hyunsuk Yang, Kyung Mi Park, Hansoo Park. CNTN4 is a novel immune checkpoint protein that inhibits proliferation of T cell by interacting with APP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 492.

Antisense Oligonucleotide-Induced Amyloid Precursor Protein Splicing Modulation as a Therapeutic Approach for Dutch-Type Cerebral Amyloid Angiopathy.

Dutch-type cerebral amyloid angiopathy (D-CAA) is a monogenic form of cerebral amyloid angiopathy and is inherited in an autosomal dominant manner. The disease is caused by a point mutation in exon 17 of the amyloid precursor protein (APP) gene that leads to an amino acid substitution at codon 693. The mutation is located within the amyloid beta (Aβ) domain of APP, and leads to accumulation of toxic Aβ peptide in and around the cerebral vasculature. We have designed an antisense oligonucleotide (AON) approach that results in skipping of exon 17, generating a shorter APP isoform that lacks part of the Aβ domain and the D-CAA mutation. We demonstrate efficient AON-induced skipping of exon 17 at RNA level and the occurrence of a shorter APP protein isoform in three different cell types. This resulted in a reduction of Aβ40 in neuronally differentiated, patient-derived induced pluripotent stem cells. AON-treated wild-type mice showed successful exon skipping on RNA and protein levels throughout the brain. These results illustrate APP splice modulation as a promising therapeutic approach for D-CAA.

Altered Hinge Conformations in APP Transmembrane Helix Mutants May Affect Enzyme-Substrate Interactions of γ-Secretase.

Cleavage of substrates by γ-secretase is an inherently slow process where substrate-enzyme affinities cannot be broken down into specific sequence requirements in contrast to soluble proteases. Nevertheless, despite its apparent sequence tolerance single point mutations in amyloid precursor protein can severely affect cleavage efficiencies and change product line preferences. We have determined by NMR spectroscopy the structures of the transmembrane domain of amyloid precursor protein in TFE/water and compared it to that of four mutants: two FAD mutants, V44M and I45T, and the two diglycine hinge mutants, G38L and G38P. In accordance with previous publications, the transmembrane domain is composed of two helical segments connected by the diglycine hinge. Mutations alter kink angles and structural flexibility. Furthermore, to our surprise, we observe different, but specific mutual orientations of N- and C-terminal helical segments in the four mutants compared to the wildtype. We speculate that the observed orientations for G38L, G38P, V44M, and I45T lead to unfavorable interactions with γ-secretase exosites during substrate movement to the enzyme's active site in presenilin and/or for the accommodation into the substrate-binding cavity of presenilin.

Impact of Cholesterol Concentration and Lipid Phase on Structure and Fluctuation of Amyloid Precursor Protein.

Elevated levels of cellular cholesterol have been identified as one factor contributing to the onset of Alzheimer's disease (AD). Specific interaction between cholesterol and the amyloid precursor protein (APP), investigated via NMR experiments and computational studies, has been proposed to play a critical role in the processing of APP by secretases and the biogenesis of amyloid-β (Aβ) protein. We present all-atom molecular dynamics simulations of the 40-residue congener of the C-terminal domain of APP, C9916-55 (C99), in cholesterol-enriched DMPC lipid bilayers. We investigated the effect of cholesterol concentration on the conformational ensemble of wild-type C99 and C99-cholesterol associations at the low pH of endosomal environments, at which residues E22 and D23 are neutral. C99 was also characterized in liquid ordered domains for Dutch (E22Q) and Iowa (D23N) Familial AD mutants at low pH and for the wild-type sequence using protonation states characteristic of neutral pH. Our results reproduce the equilibrium constant of past NMR characterizations of the C99-cholesterol interaction but are not consistent with the C99-cholesterol binding hypothesis. We find that the lifetimes of both DMPC and cholesterol complexed with C99 display a power-law distribution of residence lifetimes. Longer-lived C99-DMPC and C99-cholesterol complexes are primarily stabilized by salt bridges and hydrogen bonds of lysine amines to phosphate and hydroxyl groups. Nevertheless, specific interfaces for C99-cholesterol association which are not present for DMPC can be identified. Changes to C99-cholesterol interfaces are found to depend on C99 tilt angle and orientation of the juxtamembrane domain of C99 containing residues E22 and D23. These observations support a more nuanced view of the C99-cholesterol interaction than has previously been suggested. We propose that cholesterol modulates the conformation and activity of C99 and other small transmembrane proteins indirectly through induction of the liquid ordered phase and directly through hydrogen bonding. This suggests a critical role for membrane heterogeneity introduced by cholesterol in modulating the structural ensemble of C99 and the production of Aβ.

Brain connectivity and activity during Alzheimer’s disease progression in a mouse model by manganese‐‐enhanced MRI in living brain correlated with post‐mortem histopathology.

Alzheimer’s disease (AD) arises from inherited mutations in genes associated with plaque formation: amyloid precursor protein (APP), tau, and presenilin, or sporadically without these mutations. Studies of mice expressing these familial Alzheimer’s disease (FAD)‐associated mutations has led to new insights into the biological processes in sporadic AD. We study a mouse model in which expression of the FAD protein, APPSwInd, can be suppressed after plaque formation. This mouse thus corresponds to sporadic Alzheimer’s disease, where plaques are present in the absence of mutated protein. We use manganese‐enhanced magnetic resonance imaging (MEMRI), an emerging technology, coupled with histopathology of serial sections to investigate how plaques affect connectivity, neural activity degeneration throughout the whole brain as the mouse ages. MEMRI can be used in two ways: to determine axonal transport dynamics and to map neural activity in living mice. Stereotactic intracranial injection of Mn2+ traces transport in hippocampus occurring over 24h in the awake behaving mouse. Systemic Mn2+ enters active neurons in the brain of freely moving mice over 24h and is imaged retrospectively. Amyloid precursor protein (APP) is the precursor to A□eta plaques. The cytoplasmic domain of APP mediates cargo‐motor attachments for axonal transport. Our results show that in APP‐KO mice, axonal transport on Mn2+ is decreased demonstrating the APP plays a role in Mn2+ transport. In old transgenic mice APPSwInd, with both over‐expression of mutated protein and plaques, axonal transport is altered, as detected by time‐lapse manganese‐enhanced magnetic resonance imaging (MEMRI) of the brain in living mice. Time‐lapse MR images were captured before and successive time points after stereotactic injection of Mn2+ (3–5nL) into CA3 of the hippocampus. Images of multiple individuals from the two groups (with plaques and no expression, or without plaques) were aligned and processed with our automated computational pipeline, and voxel‐wise statistical parametric mapping (SPM) performed. Whole brain neural activity was decreased in 3yr old mice with plaque and expressing APPSwInd compared to non‐expressing littermates, particularly in the hippocampus. Brains were harvested after MEMRI for biochemistry, or fixed, embedded and serially sectioned for histopathology. Histopathology detected mouse p‐tau–positive dystrophic neurites, decreases in the volume of septal nuclei, decrease in dentate gyrus width and cell death. Thus, plaque alone depresses transport in the important hippocampal to basal forebrain memory circuit, and decreases neural activity in the aging mouse.Support or Funding InformationSupported by NINDS RO1 NS062184 and The Harvey Family Endowment.

Proinflammatory cytokines induce accumulation of glypican-1-derived heparan sulfate and the C-terminal fragment of β-cleaved APP in autophagosomes of dividing neuronal cells.

Proinflammatory cytokines stimulate expression of β-secretase, which increases processing of amyloid precursor protein (APP), ultimately leading to the deposition of amyloid beta (Aβ). The N-terminal domain of β-cleaved APP supports Cu/NO-dependent release of heparan sulfate (HS) from the glypican-1 (Gpc-1) proteoglycan. HS is an inhibitor of β-secretase, thereby constituting a regulatory, negative feedback loop. Here, we have investigated the effect of the proinflammatory cytokines TNF-α, IL-1β and IL-6 on the interplay between APP processing and release of HS from Gpc-1 in neuronal cells. We have used deconvolution immunofluorescence microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a panel of monoclonal/polyclonal antibodies recognizing the released HS, the N-terminus of Aβ, Aβ, the C-terminus of APP and the autophagosome marker LC3 as well as the chemical lysosome marker LysoTrackerRed (LTR). We repeatedly found that N2a neuroblastoma cells and human neural stem cells grown in the presence of the cytokines developed large cytoplasmic clusters, which stained positive for HS, the N-terminus of Aβ, Aβ, the C-terminus of APP, LC3 and LTR, indicating accumulation of HS and APP/APP degradation products in enlarged autophagosomes/lysosomes. The SDS-PAGE of immunoisolates obtained from TNF-α-treated N2a cells by using anti-C-terminus of APP revealed the presence of SDS-stable complexes between HS and the C-terminal fragment of β-cleaved APP (βCTF) migrating in the range 10–18 kDa. Clustered accumulation of βCTF disappeared when HS release was prevented and slightly enhanced when HS release was increased. Hence, when proinflammatory cytokines induce increased processing of APP, inhibition of β-secretase by HS is insufficient, which may lead to the impaired autophagosomal degradation.

Defining the binding interface of Amyloid Precursor Protein (APP) and Contactin3 (CNTN3) by site-directed mutagenesis

The Amyloid Precursor Protein (APP) and Contactin (CNTN) families of cell-surface proteins have been intensively studied in the context of neural development and neuropsychiatric diseases. Earlier studies demonstrated both genetic and biochemical interactions between the extracellular domains of APP and CNTN3, but their precise binding interfaces were not defined. In the present study, we have used binding assays between APP-alkaline phosphatase (AP) fusion proteins and CNTN-Fc fusion proteins, together with alanine substitution mutagenesis, to show that: (i) the second Fibronectin domain (Fn(2)) in CNTN3 mediates APP binding; (ii) the copper binding domain (CuBD) in APP mediates CNTN3 binding; and (iii) the most important amino acids for APP-CNTN3 binding reside on one face of CNTN3-Fn(2) and on one face of APP-CuBD. These experiments define the regions of direct contact that mediate the binding interaction between APP and CNTN3.