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Nonlinear frequency division multiplexing (NFDM), as a possible technique to overcome the limit imposed by Kerr nonlinearity in conventional coherent optical communication systems, has attracted widespread attention in the communication community in recent years. In order to fully utilize the available degrees of freedom in the nonlinear spectrum, this paper focuses on the full-spectrum (FS) modulated NFDM system. First, we maximize the data rate of discrete spectrum (DS) by optimizing the distribution of eigenvalues in DS part of FS. Then through introducing the probabilistic shaping (PS) into the FS system, and combined with linear minimum mean square (LMMSE) estimators, a 1120 km transmission with BER below the hard decision forward error correction (HD-FEC) threshold at 112 Gbps is achieved, where 128 subcarriers with PS-64QAM are used in the continuous spectrum (CS) and 13 eigenvalues with 64QAM are adopted in the discrete spectrum (DS). The achievable data rate is about 12% higher than that of pure CS modulation. Our work achieves the current FS NFDM system with the largest number of multiplexed eigenvalues, and provides a way to improve the performance of FS systems.

We demonstrate, for the first time, a record 113 Gb/s (spectral efficiency of 2.8 bits/s/Hz) full-spectrum modulated nonlinear frequency division transmission system with 256 nonlinear subcarriers for continuous spectrum and 11 eigenvalues for discrete spectrum.

In optical fiber communication systems, the tendency to prefer increasingly higher baud rates, up to 100 or 200 Gbaud, compels us to deal with wideband fiber channels in which polarization effects must be treated in a more complicated manner. In contrast to the conventional 50 GHz bandwidth wavelength division multiplexing channel, polarization mode dispersion (PMD) is observed in an all-order manner in a wideband fiber channel beyond 100 GHz, rather than only first- and second-order. In this study, certain principles that should be followed in establishing a PMD emulation model in a wideband fiber channel were put forward. Then, a temporal and spectral evolution PMD model that can reflect and emulate the evolution features of the polarization effects in a real fiber channel was proposed and demonstrated. The analyses show the PMD model satisfies all the principles for the wideband channels. Finally, we suggested some approaches to define a narrowband or wideband channel only considering the polarization effects to deal with PMD in a high baud rate optical fiber communication system.

Polarization mode dispersion (PMD) is one of the fundamental properties of a standard single-mode fiber. It affects the propagating signals and degrades the performance of high-speed optical fiber communication systems. PMD also gives an effect on the nonlinear spectra or scattering data in nonlinear frequency division multiplexing (NFDM) systems. However, PMD is usually described in the linear frequency domain, and there are few investigations about the influence of PMD in the nonlinear frequency domain (NFD). An NFD-PMD model is needed to understand the impact of PMD in the NFD. In this work, using a linear approximation method, we first propose an NFD-PMD model and verify its effectiveness. With the guide of the NFD-PMD model, a blind NFD-PMD equalization scheme is designed. The simulation results indicate that the proposed NFD-PMD equalization scheme has better performance than the training sequence method based on linear frequency domain equalization.

Modulation format identification (MFI) is crucial in next-generation optical networks such as cognitive optical networks. An integrated-clustering-algorithm-based MFI scheme in a coherent optical communication system is proposed herein. Numerical simulations are performed to test the performance of the scheme on the platform of a polarization domain multiplexing system at a symbol rate of 28 Gbaud. Simulations show that the MFI scheme can achieve an accuracy of 100% for five modulation formats considered in this study when the optical signal-to-noise ratio values are lower than the 7% forward error correction threshold. Tolerances to residual chromatic dispersion (CD) are discussed, and the results show that the proposed scheme can perform well as the residual CD changes over a wide range of values. Moreover, by selecting a number of key blocks, the proposed scheme is less complex than other clustering-based MFI schemes.

A joint scheme introducing probabilistic shaping (PS) at the transmitter and utilizing a neural network (NN) equalizer at the receiver is proposed to improve the performance of the b -modulated nonlinear frequency division multiplexing (NFDM) system. Through a numerical simulation, we demonstrate that PS plays a leading role for low launch power case, which improves the performance of the system effectively, while the NN equalizer’s superiority appears in a high launch power region, whose main role is to weaken the correlation among subcarriers for improving system performance. The proposed scheme would enlighten the optimum modulation and detection schemes of the NFDM system.

An antibiotic cocktail leads to reductions in brain amyloidosis, altered morphology, and gene expression in microglia of male but not female transgenic mice that exhibit Aβ deposits. The microbiome plays a causal role in modulating Aβ burden and microglia phenotypes., We demonstrated that an antibiotic cocktail (ABX)-perturbed gut microbiome is associated with reduced amyloid-β (Aβ) plaque pathology and astrogliosis in the male amyloid precursor protein (APP)SWE/presenilin 1 (PS1)ΔE9 transgenic model of Aβ amyloidosis. We now show that in an independent, aggressive APPSWE/PS1L166P (APPPS1-21) mouse model of Aβ amyloidosis, an ABX-perturbed gut microbiome is associated with a reduction in Aβ pathology and alterations in microglial morphology, thus establishing the generality of the phenomenon. Most importantly, these latter alterations occur only in brains of male mice, not in the brains of female mice. Furthermore, ABX treatment lead to alterations in levels of selected microglial expressed transcripts indicative of the “M0” homeostatic state in male but not in female mice. Finally, we found that transplants of fecal microbiota from age-matched APPPS1-21 male mice into ABX-treated APPPS1-21 male restores the gut microbiome and partially restores Aβ pathology and microglial morphology, thus demonstrating a causal role of the microbiome in the modulation of Aβ amyloidosis and microglial physiology in mouse models of Aβ amyloidosis.

The study demonstrates that the S198P mutation in APP promotes accelerated folding and egress to late compartments leading to enhanced Aβ production and deposition in vivo., Familial Alzheimer’s disease (FAD)–linked mutations in the APP gene occur either within the Aβ-coding region or immediately proximal and are located in exons 16 and 17, which encode Aβ peptides. We have identified an extremely rare, partially penetrant, single nucleotide variant (SNV), rs145081708, in APP that corresponds to a Ser198Pro substitution in exon 5. We now report that in stably transfected cells, expression of APP harboring the S198P mutation (APPS198P) leads to elevated production of Aβ peptides by an unconventional mechanism in which the folding and exit of APPS198P from the endoplasmic reticulum is accelerated. More importantly, coexpression of APP S198P and the FAD-linked PS1ΔE9 variant in the brains of male and female transgenic mice leads to elevated steady-state Aβ peptide levels and acceleration of Aβ deposition compared with age- and gender-matched mice expressing APP and PS1ΔE9. This is the first AD-linked mutation in APP present outside of exons 16 and 17 that enhances Aβ production and deposition.

A combination scheme of 4D-2A8PSK modulation with neural network receiver is proposed to improve the performance of the discrete spectra modulated nonlinear frequency division multiplexing systems. The transmission distance extends from 1000km to 2100km.

The parameters in the waveform shaping stage of the transmitter are critical to the performance of the NFDM system. By designing and optimizing these parameters, the performance of the NFDM system can be effectively improved. © 2021 The Author(s)

A nonlinear frequency division multiplexing (NFDM) transmission system, designed specifically for nonlinear fiber channel, has the potential to overcome the nonlinear Shannon capacity limit. However, the spectral efficiency (SE) of the current proven NFDM transmission systems is still lower than that of the analogous orthogonal frequency division multiplexing system. It is extremely necessary to explore effective modulation scheme for the aim of increasing the SE of NFDM system. In this study, we first propose the nonlinear-frequency-packing nonlinear frequency division multiplexing (NFP-NFDM) transmission system. In NFP-NFDM, the spacing of nonlinear subcarriers is squeezed and more nonlinear subcarriers can be packed, but the inter carrier interference (ICI) is introduced. The method of NFP in nonlinear Fourier domain is carefully designed to reduce the complexity of ICI cancellation. Through numerical simulation, we illustrate the feasibility of NFP-NFDM transmission, and higher SE in NFP-NFDM than that of NFDM system is also demonstrated. The upper bound of the normalized SE for NFP-NFDM is estimated, which is higher than that of current NFDM system. Besides, we find out that the NFP scheme may have the advantage of reducing the signal-noise interaction in fiber transmission scenario, which indicates there may be a better way to load the data into the nonlinear Fourier domain.

We previously demonstrated that lifelong antibiotic (ABX) perturbations of the gut microbiome in male APPPS1-21 mice lead to reductions in amyloid β (Aβ) plaque pathology and altered phenotypes of plaque-associated microglia. Here, we show that a short, 7-d treatment of preweaned male mice with high-dose ABX is associated with reductions of Aβ amyloidosis, plaque-localized microglia morphologies, and Aβ-associated degenerative changes at 9 wk of age in male mice only. More importantly, fecal microbiota transplantation (FMT) from transgenic (Tg) or WT male donors into ABX-treated male mice completely restored Aβ amyloidosis, plaque-localized microglia morphologies, and Aβ-associated degenerative changes. Transcriptomic studies revealed significant differences between vehicle versus ABX-treated male mice and FMT from Tg mice into ABX-treated mice largely restored the transcriptome profiles to that of the Tg donor animals. Finally, colony-stimulating factor 1 receptor (CSF1R) inhibitor-mediated depletion of microglia in ABX-treated male mice failed to reduce cerebral Aβ amyloidosis. Thus, microglia play a critical role in driving gut microbiome–mediated alterations of cerebral Aβ deposition.

We introduce probabilistic shaping at the transmitter and apply neural network spectrum equalizer at the receiver in 64-QAM NFDM system. The proposed scheme possesses higher mutual information and further transmission distance. © 2020 The Author(s)

A nonlinear Fourier domain PMD impairment model is first proposed and verified. It can help to better understand the effects of PMD, and provide guidance for the design of corresponding equalization scheme. © 2020 The Author(s)

γ-secretase is a macromolecular complex that catalyzes intramembranous hydrolysis of more than 100 membrane-bound substrates. The complex is composed of presenilin (PS1 or PS2), anterior pharynx defect-1 (APH-1), nicastrin (NCT) and PEN-2 and early-onset; autosomal dominant forms of Alzheimer’s disease (AD) are caused by inheritance of mutations of PS. No mutations in genes encoding NCT, or PEN-2 have been identified to date that cause AD. In this regard, a large genetic meta-analysis of four cohorts consisting of more than 600 000 individuals identified a common missense variant, rs117618017 in the APH1B gene that results in a T27I mutation, as a novel genome-wide significant locus. In order to confirm the findings that rs117618017 is associated with risk of AD, we performed a genetic screen from deep whole genome sequencing of the large NIMH family-based Alzheimer’s Disease (AD) dataset. In parallel, we sought to uncover potential molecular mechanism(s) by which APH-1B T27I might be associated with AD by generating stable HEK293 cell lines, wherein endogenous APH-1A and APH-1B expression was silenced and into which either the wild type APH-1B or the APH-1B T27I variant was stably expressed. We then tested the impact of expressing either the wild type APH-1B or the APH-1B T27I variant on γ-secretase processing of human APP, the murine Notch derivative mNΔE and human neuregulin-1. We now report that we fail to confirm the association of rs1047552 with AD in our cohort and that cells expressing the APH-1B T27I variant show no discernable impact on the γ-secretase processing of established substrates compared with cells expressing wild-type APH-1B.

Recent evidence suggests the commensal microbiome regulates host immunity and influences brain function; findings that have ramifications for neurodegenerative diseases. In the context of Alzheimer’s disease (AD), we previously reported that perturbations in microbial diversity induced by life-long combinatorial antibiotic (ABX) selection pressure in the APPSWE/PS1ΔE9 mouse model of amyloidosis is commensurate with reductions in amyloid-β (Aβ) plaque pathology and plaque-localised gliosis. Considering microbiota-host interactions, specifically during early post-natal development, are critical for immune- and neuro-development we now examine the impact of microbial community perturbations induced by acute ABX exposure exclusively during this period in APPSWE/PS1ΔE9 mice. We show that early post-natal (P) ABX treatment (P14-P21) results in long-term alterations of gut microbial genera (predominantly Lachnospiraceae and S24-7) and reduction in brain Aβ deposition in aged APPSWE/PS1ΔE9 mice. These mice exhibit elevated levels of blood- and brain-resident Foxp3+ T-regulatory cells and display an alteration in the inflammatory milieu of the serum and cerebrospinal fluid. Finally, we confirm that plaque-localised microglia and astrocytes are reduced in ABX-exposed mice. These findings suggest that ABX-induced microbial diversity perturbations during post-natal stages of development coincide with altered host immunity mechanisms and amyloidosis in a murine model of AD.

Although fruitful studies have been conducted on carrier frequency offset (CFO) estimations in linear coherent optical fiber communication systems, there are few studies on CFO estimations and recoveries in the systems based on the nonlinear Fourier transform (NFT). Although the CFO is originated from the linear frequency domain, it definitely has effects on nonlinear spectra, including the shift of the nonlinear frequency and the phase rotations of the scattering data, which are similar to its effects on linear spectra. This work indicates that it is feasible to estimate frequency offset (FO) by capturing symbol variations in the nonlinear frequency domain (NFD) rather than in the linear frequency domain; the latter was usually exploited in the literature. Based on a thorough investigation of the FO induced behavior that appears in a nonlinear frequency division multiplexing (NFDM) system, we proposed a nonlinear frequency domain estimation method aided by training symbols (TS) using an angle search algorithm after NFT operations at the receiver. The discussions in this paper prove that the proposed method is generally applicable to the NFDM systems regardless of whether using single or multiple eigenvalues. A performance comparison between the NFD method and the conventional method in the linear frequency domain is performed with different modulation formats for both single and multiple eigenvalue NFDM transmission systems. The analysis results show that the proposed method holds the better stability and estimation accuracy in contrast with the linear domain estimation method. The TS overhead can also be deduced dramatically, which implies better transmission efficiency. Therefore, the NFD method is more powerful for eigenvalue NFDM transmission systems, especially for the scenarios where high order modulation formats and multiple eigenvalues are utilized.

For nonlinear frequency division multiplexing system, a transmission model in the presence of phase noise was derived, and a blind phase noise estimation method based on decision directed free technology was exploited and verified.

Nonlinear frequency division multiplexing (NFDM) has been shown to be promising in overcoming the fiber Kerr nonlinearity limit. In multiple-eigenvalue modulated NFDM systems, the transmission capacity increases with the number of modulated eigenvalues. However, as the number of modulated eigenvalues increases, the complexities of the signal waveform and the nonlinear Fourier transform (NFT) algorithm for demodulation increase dramatically as well, while the accuracy drops significantly. Meanwhile, impairments such as amplifier spontaneous emission noise and phase noise in practical channels would perturb the eigenvalues and the corresponding nonlinear spectra during transmission. Coupled with an increase in the modulation format order, it is difficult for NFT algorithm-based receivers to recover information. To enable the use of multiple-eigenvalue modulated NFDM systems, we propose an innovative receiver based on regression neural networks (NNs), which can demodulate information correctly for both single- and dual-polarization NFDM systems. The results show that it has strong robustness and has a certain tolerance to the impairments of communication systems. In the contrast that the poor demodulation performance of the NFT and the Euclidean minimum distance (MD) receivers for multi-eigenvalue modulated NFDM systems, our proposed NN receiver can achieve low bit error rate with 2 GBaud 16QAM modulation over 1,000 km transmission in four-eigenvalue modulated single-polarization NFDM systems. The performance of three receivers (NFT, MD and NN) in a two-eigenvalue modulated NFDM system are also compared, the NN receiver shows the best performance and appears more suitable for higher-order modulation formats.

We proposed a frequency offset estimation method based on spectrum searching in discrete nonlinear frequency domain, added by training symbols. Estimation error lower than 1MHz range from ±1GHz, insensitive to linewidth of laser, was achieved.

The γ-secretase complex, composed of presenilin, nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), is assembled in a highly regulated manner and catalyzes the intramembranous proteolysis of many type I membrane proteins, including Notch and amyloid precursor protein. The Notch family of receptors plays important roles in cell fate specification during development and in adult tissues, and aberrant hyperactive Notch signaling causes some forms of cancer. γ-Secretase-mediated processing of Notch at the cell surface results in the generation of the Notch intracellular domain, which associates with several transcriptional coactivators involved in nuclear signaling events. On the other hand, γ-secretase-mediated processing of amyloid precursor protein leads to the production of amyloid β (Aβ) peptides that play an important role in the pathogenesis of Alzheimer disease. We used a phage display approach to identify synthetic antibodies that specifically target NCT and expressed them in the single-chain variable fragment (scFv) format in mammalian cells. We show that expression of a NCT-specific scFv clone, G9, in HEK293 cells decreased the production of the Notch intracellular domain but not the production of amyloid β peptides that occurs in endosomal and recycling compartments. Biochemical studies revealed that scFvG9 impairs the maturation of NCT by associating with immature forms of NCT and, consequently, prevents its association with the other components of the γ-secretase complex, leading to degradation of these molecules. The reduced cell surface levels of mature γ-secretase complexes, in turn, compromise the intramembranous processing of Notch.

Recent evidence suggests the commensal microbiome regulates host immunity and influences brain function; findings that have ramifications for neurodegenerative diseases. In the context of Alzheimer’s disease (AD), we previously reported that perturbations in microbial diversity induced by life-long combinatorial antibiotic (ABX) selection pressure in the APPSWE/PS1ΔE9 mouse model of amyloidosis is commensurate with reductions in amyloid-β (Aβ) plaque pathology and plaque-localised gliosis. Considering microbiota-host interactions, specifically during early post-natal development, are critical for immune- and neuro-development we now examine the impact of microbial community perturbations induced by acute ABX exposure exclusively during this period in APPSWE/PS1ΔE9 mice. We show that early post-natal (P) ABX treatment (P14-P21) results in long-term alterations of gut microbial genera (predominantly Lachnospiraceae and S24-7) and reduction in brain Aβ deposition in aged APPSWE/PS1ΔE9 mice. These mice exhibit elevated levels of blood- and brain-resident Foxp3+ T-regulatory cells and display an alteration in the inflammatory milieu of the serum and cerebrospinal fluid. Finally, we confirm that plaque-localised microglia and astrocytes are reduced in ABX-exposed mice. These findings suggest that ABX-induced microbial diversity perturbations during post-natal stages of development coincide with altered host immunity mechanisms and amyloidosis in a murine model of AD.

Alzheimer's disease (AD) is characterized pathologically by an abundance of extracellular neuritic plaques composed primarily of the 42-amino acid amyloid β peptide variant (Aβ42). In the majority of familial AD (FAD) cases, e.g., those harboring mutations in presenilin 1 (PS1), there is a relative increase in the levels of Aβ42 compared to the levels of Aβ40. We previously reported the characterization of a series of aminothiazole-bridged aromates termed aryl aminothiazole γ-secretase modulators or AGSMs [Kounnas, M. Z., et al. (2010) Neuron 67, 769-780] and showed their potential for use in the treatment of FAD [Wagner, S. L., et al. (2012) Arch. Neurol. 69, 1255-1258]. Here we describe a series of GSMs with physicochemical properties improved compared to those of AGSMs. Specific heterocycle replacements of the phenyl rings in AGSMs provided potent molecules with improved aqueous solubilities. A number of these soluble γ-secretase modulators (SGSMs) potently lowered Aβ42 levels without inhibiting proteolysis of Notch or causing accumulation of amyloid precursor protein carboxy-terminal fragments, even at concentrations approximately 1000-fold greater than their IC50 values for reducing Aβ42 levels. The effects of one potent SGSM on Aβ peptide production were verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, showing enhanced production of a number of carboxy-truncated Aβ species. This SGSM also inhibited Aβ42 peptide production in a highly purified reconstituted γ-secretase in vitro assay system and retained the ability to modulate γ-secretase-mediated proteolysis in a stably transfected cell culture model overexpressing a human PS1 mutation validating the potential for use in FAD.

Presenilins 1 and 2 (PS1 and PS2) are the catalytic subunits of the γ-secretase complex, and genes encoding mutant PS1 and PS2 variants cause familial forms of Alzheimer's disease. Lee et al. (2010) recently reported that loss of PS1 activity lead to impairments in autophagosomal function as a consequence of lysosomal alkalinization, caused by failed maturation of the proton translocating V0a1 subunit of the vacuolar (H+)-ATPase and targeting to the lysosome. We have reexamined these issues in mammalian cells and in brains of mice lacking PS (PScdko) and have been unable to find evidence that the turnover of autophagic substrates, vesicle pH, V0a1 maturation, or lysosome function is altered compared with wild-type counterparts. Collectively, our studies fail to document a role for presenilins in regulating cellular autophagosomal function. On the other hand, our transcriptome studies of PScdko mouse brains reveal, for the first time, a role for PS in regulating lysosomal biogenesis.

The γ-secretase complex, composed of presenilin, anterior-pharynx-defective 1, nicastrin, and presenilin enhancer 2, catalyzes the intramembranous processing of a wide variety of type I membrane proteins, including amyloid precursor protein (APP) and Notch. Earlier studies have revealed that nicastrin, a type I membrane-anchored glycoprotein, plays a role in γ-secretase assembly and trafficking and has been proposed to bind substrates. To gain more insights regarding nicastrin structure and function, we generated a conformation-specific synthetic antibody and used it as a molecular probe to map functional domains within nicastrin ectodomain. The antibody bound to a conformational epitope within a nicastrin segment encompassing residues 245–630 and inhibited the processing of APP and Notch substrates in in vitro γ-secretase activity assays, suggesting that a functional domain pertinent to γ-secretase activity resides within this region. Epitope mapping and database searches revealed the presence of a structured segment, located downstream of the previously identified DAP domain ( D YIGS a nd p eptidase; residues 261–502), that is homologous to a tetratricopeptide repeat (TPR) domain commonly involved in peptide recognition. Mutagenesis analyses within the predicted TPR-like domain showed that disruption of the signature helical structure resulted in the loss of γ-secretase activity but not the assembly of the γ-secretase and that Leu571 within the TPR-like domain plays an important role in mediating substrate binding. Taken together, these studies offer provocative insights pertaining to the structural basis for nicastrin function as a “substrate receptor” within the γ-secretase complex.

A complex composed of presenilin (PS), nicastrin, PEN-2, and APH-1 is absolutely required for γ-secretase activity in vivo. Evidence has emerged to suggest a role for PS as the catalytic subunit of γ-secretase, but it has not been established that PS is catalytically active in the absence of associated subunits. We now report that bacterially synthesized, recombinant PS (rPS) reconstituted into liposomes exhibits γ-secretase activity. Moreover, an rPS mutant that lacks a catalytic aspartate residue neither exhibits reconstituted γ-secretase activity nor interacts with a transition-state γ-secretase inhibitor. Importantly, we demonstrate that rPS harboring mutations that cause early onset familial Alzheimer’s disease (FAD) lead to elevations in the ratio of Aβ42 to Aβ40 peptides produced from a wild-type APP substrate and that rPS enhances the Aβ 42/ Aβ 40 peptide ratio from FAD-linked mutant APP substrates, findings that are entirely consistent with the results obtained in in vivo settings. Thus, γ-secretase cleavage specificity is an inherent property of the polypeptide. Finally, we demonstrate that PEN2 is sufficient to promote the endoproteolysis of PS1 to generate the active form of γ-secretase. Thus, we conclusively establish that activated PS is catalytically competent and the bimolecular interaction of PS1 and PEN2 can convert the PS1 zymogen to an active protease.

Cerebral deposition of beta-amyloid (Abeta) peptides is a pathological hallmark of Alzheimer disease. Intramembranous proteolysis of amyloid precursor protein by a multiprotein gamma-secretase complex generates Abeta. Previously, it was reported that CD147, a glycoprotein that stimulates production of matrix metalloproteinases (MMPs), is a subunit of gamma-secretase and that the levels of secreted Abeta inversely correlate with CD147 expression. Here, we show that the levels and localization of CD147 in fibroblasts, as well as postnatal expression and distribution in brain, are distinct from those of integral gamma-secretase subunits. Notably, we show that although depletion of CD147 increased extracellular Abeta levels in intact cells, membranes isolated from CD147-depleted cells failed to elevate Abeta production in an in vitro gamma-secretase assay. Consistent with an extracellular source that modulates Abeta metabolism, synthetic Abeta was degraded more rapidly in the conditioned medium of cells overexpressing CD147. Moreover, modulation of CD147 expression had no effect on epsilon-site cleavage of amyloid precursor protein and Notch1 receptor. Collectively, our results demonstrate that CD147 modulates Abeta levels not by regulating gamma-secretase activity, but by stimulating extracellular degradation of Abeta. In view of the known function of CD147 in MMP production, we postulate that CD147 expression influences Abeta levels by an indirect mechanism involving MMPs that can degrade extracellular Abeta.

Understanding of the structure of the γ-secretase complex consisting of presenilin (PS), anterior pharynx-defective 1 (APH-1), nicastrin (NCT), and presenilin enhancer 2 (PEN-2) is of significant therapeutic interest for the design of γ-secretase modulators for Alzheimer disease. The structure of γ-secretase revealed by cryo-EM approaches suggested a substrate binding mechanism for NCT, a bilobar structure that involved rotation of the two lobes around a central pivot and opening of a “lid” region that facilitates substrate recruitment. To validate this proposal, we expressed NCT that lacks the lid entirely, or a variety of NCT variants that harbor mutations at highly conserved residues in the lid region in NCT-deficient cells, and then assessed their impact on γ-secretase assembly, activity, and stability. In addition, we assessed the impact of mutating a critical residue proposed to be a pivot around which the two lobes of NCT rotate. Our results show that neither the mutations on the lid tested here nor the entire lid deletion has any significant impact on γ-secretase assembly, activity, and stability, and that NCT with the mutation of the proposed pivot rescues γ-secretase activity in NCT-deficient cells in a manner indistinguishable from WT NCT. These findings indicate that the NCT lid is not an essential element necessary for γ-secretase assembly, activity, and stability, and that rotation of the two lobes appears not to be a prerequisite for substrate binding and γ-secretase function.

The green fluorescent protein (GFP) has become a powerful tool in molecular and cell biology. It is a commonly used marker for cloning and transfection experiments as well as a useful label of living cells allowing continuous observation of developing structures. In order to unravel mechanisms of neuronal differentiation, we generated a transgenic mouse model which expresses GFPS65T,hu under the control of the Purkinje cell-specific promoter L7/pcp-2. Here, we show that GFPS65T,hu is highly expressed specifically in the cerebellum in whole mount preparations after the 2nd postnatal week. GFPS65T,hu can be detected exclusively in Purkinje cells of cerebellar slices. The fluorescence intensity of GFPS65T,hu should enable the characterization and recording of axons, dendrites, and spines protruding from these neuronal processes. The level of GFP expression can be quantified by western blotting which allows to analyze protein expression and L7/pcp-2 promoter regulation in vivo. The application of cellular and physiological techniques on L7GFP mice will provide a remarkable opportunity to investigate various aspects of neuronal development at the cellular and subcellular levels.

We have previously demonstrated that a short sequence element (L7ATE) within the proximal promoter of a Purkinje cell-specific gene, pcp-2(L7), is required for the normal pattern of expression of the gene in the cerebellum of transgenic mice. The presence of a series of TAAT sequence motifs in this element suggested its interaction with homeodomain proteins. To extend these observations, degenerate oligonucleotides were used to clone by reverse-transcriptase polymerase chain reaction members of the mouse Hox gene family expressed in neonatal cerebellum but not forebrain. Two of these, HoxB7 and HoxA5, are continuously expressed from the neonatal period into adult stages in cerebellar Purkinje cells. These Hox proteins are shown to synergistically activate the L7 promoter by cotransfection assay in vitro. In contrast, another homeodomain protein that is normally expressed in Purkinje cells only during the embryonic period, En-2, has a negative effect on L7 gene expression. These data suggest a biphasic, combinatorial control mechanism for the Purkinje cell-specific expression of the pcp-2(L7) gene.

Alzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta(42) levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta(40) levels while concomitantly elevating levels of Abeta(38) and Abeta(37). Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta(42); chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g., intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).

Pcp2(L7) is a Purkinje cell-specific GoLoco domain protein that modulates activation of Galphai/o proteins by G protein-coupled receptors. A likely downstream effector of this pathway is the P-type Ca(2+) channel, and thereby, the intrinsic electrophysiology of Purkinje cells could be modulated by Pcp2(L7). It has long been known that the Pcp2(L7) mRNA is abundantly localized in dendrites, suggesting the possibility of distal synthesis and local changes in levels of the protein. As a first step to uncover the trafficking and translational mechanisms for this mRNA, we have begun identifying the cis-acting sequences important for its localization in dendrites. Using expression of modified transgenes in vivo, we show that the 3'UTR, only 65 bases long, is necessary in this process.

The integral membrane protein complex gamma-secretase catalyzes the final step in the production of amyloid beta-peptides involved in the pathogenesis of Alzheimer's disease. Using negative-stain single-particle electron microscopy we have determined the structure of a native-like 500kDa gamma-secretase complex comprising presenilin, nicastrin, APH-1, and PEN-2 that is fully catalytically active. Antibody labeling of the extracellular domain of nicastrin was employed to ascertain the topology of the reconstruction. Active site labeling with a gold-coupled transition state analog inhibitor demonstrates that gamma-secretase contains a single active site facing a large conical internal cavity. This cavity, surrounded by a ∼35A thick transmembrane protein wall, extends from the extracellular side of the membrane to past the membrane centre, where it narrows to finally close at the cytoplasmic side. Based on our structure we suggest a model for gamma-secretase function, in which a hydrophobic transmembrane helix substrate is hydrolyzed by catalytic aspartyl moieties at the interface of a water-accessible internal cavity away from the surrounding lipid environment.

Background The γ-secretase complex, composed of transmembrane proteins termed presenilin (PS), anterior pharynx defective (APH), nicastrin (NCT), and presenilin enhancer-2 (Pen-2) catalyzes intramembranous hydrolysis of a variety of Type I membrane protein substrates. In order to understand aspects of subunit assembly, interactions, dynamics and catalysis, it is essential to clarify the membrane topology of each polypeptide. Hydophathicity plots predict that the 101 amino acid Pen-2 molecule has two hydrophobic domains (HP1 and HP2) that may serve as transmembrane spanning domains. Earlier reports indicated that transiently overexpressed Pen-2 uses these two hydrophobic domains as transmembrane helices that generates a “U-shaped” hairpin topology with both amino- (N-) and carboxyl-(C-) termini facing the lumen. In this report, we have reexamined the topology of endogenous Pen-2 and Pen-2 chimeras that are stably expressed in mammalian cells, and have assessed the function of these molecules in rescuing γ-secretase activity in Pen-2-deficient fibroblasts. Results We confirm that the Pen-2 C-terminus is lumenal, but the N-terminus of Pen-2 is exposed to the cytoplasm, thus indicating that HP1 does not traverse the lipid bilayer as a transmembrane domain. Domain swapping studies reveal the importance of specific regions within the first hydrophobic domain of Pen-2 that are critical for generating the topology that is a prerequisite for mediating PS1 endoproteolysis and γ-secretase activity. Finally, we report that the first fourteen amino acids of the Pen-2 HP1 are required for γ-secretase activity. Conclusions We propose that the first hydrophobic domain of Pen-2 forms a structure similar to a reentrant loop while the second hydrophobic domain spans the lipid bilayer.