Enhanced amyloid-ß generation by γ-secretase complex in DRM microdomains with reduced cholesterol levels.

A neuropathologic hallmark of Alzheimer's disease (AD) is the presence of senile plaques that contain neurotoxic amyloid-ß protein (Aß) species, which are generated by the cleavage of amyloid ß-protein precursor by secretases such as the γ-secretase complex, preferentially located in detergent-resistant membrane (DRM) regions and comprising endoproteolysed amino- and carboxy-terminal fragments of presenilin, nicastrin, anterior pharynx defective 1 and presenilin enhancer 2. Whereas some of familial AD patients harbor causative PSEN mutations that lead to more generation of neurotoxic Aß42, the contribution of Aß generation to sporadic/late-onset AD remains unclear. We found that the carboxy-terminal fragment of presenilin 1 was redistributed from DRM regions to detergent-soluble membrane (non-DRM) regions in brain tissue samples from individuals with sporadic AD. DRM fractions from AD brain sample had the ability to generate significantly more Aß and had a lower cholesterol content than DRM fractions from non-demented control subjects. We further demonstrated that lowering the cholesterol content of DRM regions from cultured cells contributed to the redistribution of γ-secretase components and Aß production. Taken together, the present analyses suggest that the lowered cholesterol content in DRM regions may be a cause of sporadic/late-onset AD by enhancing overall Aß generation.

Cytoplasmic granule formation by FUS-R495X is attributable to arginine methylation in all Gly-rich, RGG1 and RGG2 domains.

Many mutations in the fused in sarcoma (FUS) gene have been identified as genetic causative factors of amyotrophic lateral sclerosis (ALS). As a certain number of mutants form aberrant cytoplasmic granules under specific conditions, granule forming ability of FUS is believed to be linked to the pathogenesis of ALS. However, molecular mechanisms underlying this property remain unclear. An ALS-linked FUS mutant, R495X, shows extensive cytoplasmic localization and forms granules in neurons. In the present study, using R495X domain deletion constructs, we showed that deletion of any of Gly-rich, RGG1 or RGG2 significantly suppressed granule formation. Furthermore, when neurons expressing EGFP-R495X were treated with an arginine methylation inhibitor, the number of cells displaying R495X granules was significantly reduced. When FLAG-tagged arginine N-methyltransferase 8 (PRMT8) was co-expressed with EGFP-R495X to facilitate its methylation, the number of cells with granules was significantly increased. Collectively, these findings suggest that cytoplasmic granule formation by R495X is attributable to the arginine methylation in all Gly-rich, RGG1 and RGG2 domains.

X11 and X11-like proteins regulate the level of extrasynaptic glutamate receptors.

X11/Mint 1 and X11-like (X11L)/Mint 2 are neuronal adaptor protein to regulate trafficking and/or localization of various membrane proteins. By analyzing the localization of neuronal membrane proteins in X11-, X11L-, and X11/X11L doubly deficient mice with membrane fractionation procedures, we found that deficient of X11 and X11L decreased the level of glutamate receptors in non-PSD fraction. This finding suggests that X11 and X11L regulate the glutamate receptor micro-localization to the extrasynaptic region. In vitro coimmunoprecipitation studies of NMDA receptors lacking various cytoplasmic regions with X11 and X11L proteins harboring domain deletion suggest that extrasynaptic localization of NMDA receptor may be as a result of the multiple interactions of the receptor subunits with X11 and X11L regulated by protein phosphorylation, while that of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits is not dependent on the binding with X11 and X11L proteins. Because the loss of X11 and X11L tends to impair the exocytosis, but not endocytosis, of glutamate receptors, NMDA receptors are likely to be supplied to the extrasynaptic plasma membrane with a way distinct from the mechanism regulating the localization of NMDA receptors into synaptic membrane region. Reduced localization of NMDA receptor into the extrasynaptic region increased slightly the phosphorylation level of cAMP responsible element binding protein in brain of X11/X11L doubly deficient mice compare to wild-type mice, suggesting a possible role of X11 and X11L in the regulation of signal transduction pathway through extrasynaptic glutamate receptors. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

CLIPSeqTools--a novel bioinformatics CLIP-seq analysis suite.

Immunoprecipitation of RNA binding proteins (RBPs) after in vivo crosslinking, coupled with sequencing of associated RNA footprints (HITS-CLIP, CLIP-seq), is a method of choice for the identification of RNA targets and binding sites for RBPs. Compared with RNA-seq, CLIP-seq analysis is widely diverse and depending on the RBPs that are analyzed, the approaches vary significantly, necessitating the development of flexible and efficient informatics tools. In this study, we present CLIPSeqTools, a novel, highly flexible computational suite that can perform analysis from raw sequencing data with minimal user input. It contains a wide array of tools to provide an in-depth view of CLIP-seq data sets. It supports extensive customization and promotes improvization, a critical virtue, since CLIP-seq analysis is rarely well defined a priori. To highlight CLIPSeqTools capabilities, we used the suite to analyze Ago-miRNA HITS-CLIP data sets that we prepared from human brains.

Facilitation of brain mitochondrial activity by 5-aminolevulinic acid in a mouse model of Alzheimer's disease.

The activities of mitochondrial enzymes, which are essential for neural function, decline with age and in age-related disease. In particular, the activity of cytochrome c oxidase (COX/complex IV) decreases in patients with Alzheimer's disease (AD). COX, a mitochondrial inner membrane protein complex that contains heme, plays an essential role in the electron transport chain that generates ATP. Heme synthesis begins with 5-aminolevulinic acid (5-ALA) in mitochondria. 5-ALA synthetase is the rate-limiting enzyme in heme synthesis, suggesting that supplementation with 5-ALA might help preserve mitochondrial activity in the aged brain. We administered a diet containing 5-ALA to triple-transgenic AD (3xTg-AD) model mice for 6 months, starting at 3 months of age. COX activity and protein expression, as well as mitochondrial membrane potential, were significantly higher in brains of 5-ALA-fed mice than in controls. Synaptotagmin protein levels were also significantly higher in 5-ALA-fed mice, suggesting improved preservation of synapses. Although brain Aß levels tended to decrease in 5-ALA-fed mice, we observed no other significant changes in other biochemical and pathological hallmarks of AD. Nevertheless, our study suggests that daily oral administration of 5-ALA could preserve mitochondrial enzyme activities in the brains of aged individuals, thereby contributing to the preservation of neural activity.

Cytoplasmic fragment of Alcadein α generated by regulated intramembrane proteolysis enhances amyloid ß-protein precursor (APP) transport into the late secretory pathway and facilitates APP cleavage.

The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid ß-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aß generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aß in vivo.

FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns.

Dominant mutations and mislocalization or aggregation of Fused in Sarcoma (FUS), an RNA-binding protein (RBP), cause neuronal degeneration in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), two incurable neurological diseases. However, the function of FUS in neurons is not well understood. To uncover the impact of FUS in the neuronal transcriptome, we used high-throughput sequencing of immunoprecipitated and cross-linked RNA (HITS-CLIP) of FUS in human brains and mouse neurons differentiated from embryonic stem cells, coupled with RNA-seq and FUS knockdowns. We report conserved neuronal RNA targets and networks that are regulated by FUS. We find that FUS regulates splicing of genes coding for RBPs by binding to their highly conserved introns. Our findings have important implications for understanding the impact of FUS in neurodegenerative diseases and suggest that perturbations of FUS can impact the neuronal transcriptome via perturbations of RBP transcripts.

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.

A yeast functional screen predicts new candidate ALS disease genes.

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.

Membrane-microdomain localization of amyloid ß-precursor protein (APP) C-terminal fragments is regulated by phosphorylation of the cytoplasmic Thr668 residue.

Amyloid ß-precursor protein (APP) is primarily cleaved by α- or ß-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr(668) and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648-695)) with Thr(P)(668) did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aß generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aß in Alzheimer disease.

Accumulation of amyloid-ß in the brain of mouse models of Alzheimer's disease is modified by altered gene expression in the presence of human apoE isoforms during aging.

Apolipoprotein E4 (apoE4) is a risk factor for Alzheimer's disease (AD). Here, we investigated brain amyloid-ß (Aß) accumulation throughout the aging process in an amyloid precursor protein (APP) knock-in (KI) mouse model of AD that expresses human APPNL-G-F with or without human apoE4 or apoE3. Brain Aß42 levels were significantly lower in 9-month-old mice that express human isoforms of apoE than in age-matched APP-KI control mice. Linear accumulation of Aß42 began in 5-month-old apoE4 mice, and a strong increase in Aß42 levels was observed in 21-month-old apoE3 mice. Aß42 levels in cerebroventricular fluid were higher in apoE3 than in apoE4 mice at 6-7 months of age, suggesting that apoE3 is more efficient at clearing Aß42 than apoE4 at these ages. However, apoE3 protein levels were lower than apoE4 protein levels in the brains of 21-month-old apoE3 and apoE4 mice, respectively, which may explain the rapid increase in brain Aß42 burden in apoE3 mice. We identified genes that were downregulated in a human apoE-dependent (apoE4 > apoE3) and age-dependent (apoE3 = apoE4) manner, which may regulate brain Aß burden and/or AD progression. Analysis of gene expression in AD mouse models helps identify molecular mechanisms of pleiotropy by the human APOE gene during aging.

Alternative processing of γ-secretase substrates in common forms of mild cognitive impairment and Alzheimer's disease: evidence for γ-secretase dysfunction.

OBJECTIVE: The most common pathogenesis for familial Alzheimer's disease (FAD) involves misprocessing (or alternative processing) of the amyloid precursor protein (APP) by γ-secretase due to mutations of the presenilin 1 (PS1) gene. This misprocessing/alternative processing leads to an increase in the ratio of the level of a minor γ-secretase reaction product (Aß42) to that of the major reaction product (Aß40). Although no PS1 mutations are present, altered Aß42/40 ratios are also observed in sporadic Alzheimer's disease (SAD), and these altered ratios apparently reflect deposition of Aß42 as amyloid. METHODS: Using immunoprecipitation-mass spectrometry with quantitative accuracy, we analyzed in the cerebrospinal fluid (CSF) of various clinical populations the peptide products generated by processing of not only APP but also an unrelated protein, alcadein (Alc). Alc undergoes metabolism by the identical APP α-secretases and γ-secretases, yielding a fragment that we have named p3-Alc(α) because of the parallel genesis of p3-Alc(α) peptides and the p3 fragment of APP. As with Aß, both major and minor p3-Alc(α) s are generated. We studied the alternative processing of p3-Alc(α) in various clinical populations. RESULTS: We previously reported that changes in the Aß42/40 ratio showed covariance in a linear relationship with the levels of p3-Alc(α) [minor/major] ratio in media conditioned by cells expressing FAD-linked PS1 mutants. Here we studied the speciation of p3-Alc(α) in the CSF from 3 groups of human subjects (n = 158): elderly nondemented control subjects; mild cognitive impairment (MCI) subjects with a clinical dementia rating (CDR) of 0.5; SAD subjects with CDR of 1.0; and other neurological disease (OND) control subjects. The CSF minor p3-Alc(α) variant, p3-Alc(α) 38, was elevated (p < 0.05) in MCI subjects or SAD subjects, depending upon whether the data were pooled and analyzed as a single cohort or analyzed individually as 3 separate cohorts. INTERPRETATION: These results suggest that some SAD may involve alternative processing of multiple γ-secretase substrates, raising the possibility that the molecular pathogenesis of SAD might involve γ-secretase dysfunction.

A specific gene-splicing alteration in the SNRNP70 gene as a hallmark of an ALS subtype.

Amyotrophic lateral sclerosis (ALS) has been considered as one of the progressive neurodegenerative diseases. Numerous genetic factors in divergent molecular pathways have been identified as causative factors of ALS. However, the underlying molecular mechanism that causes this disease remains undetermined; as a result, this has driven the search to find consensus disease-specific hallmarks. In this study, we focused on the alteration of the ratio of two specific gene-splicing events in the SNRNP70 gene from RNA-seq data derived from patients with ALS and control subjects. The splicing profile was significantly and specifically changed in one previously identified ALS subtype. Conversely, the gene expression profile of other ALS cases containing a splicing alteration in the SNRNP70 gene was similar to that of the subtype, whereas ALS cases without this change have exhibited less similarity. These results indicate that this splicing event in the SNRNP70 gene could represent a novel and broadly applicable molecular hallmark of a subtype of ALS.

Prostaglandin E2 stimulates the production of amyloid-beta peptides through internalization of the EP4 receptor.

Amyloid-beta (Abeta) peptides, generated by the proteolysis of beta-amyloid precursor protein by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta through EP(2) and EP(4) receptors, and here we have examined the molecular mechanism. Activation of EP(2) and EP(4) receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP(2), but not EP(4), receptor-mediated stimulation of the Abeta production. In contrast, inhibitors of endocytosis suppressed EP(4), but not EP(2), receptor-mediated stimulation. Activation of gamma-secretase was observed with the activation of EP(4) receptors but not EP(2) receptors. PGE(2)-dependent internalization of the EP(4) receptor was observed, and cells expressing a mutant EP(4) receptor lacking the internalization activity did not exhibit PGE(2)-stimulated production of Abeta. A physical interaction between the EP(4) receptor and PS-1, a catalytic subunit of gamma-secretases, was revealed by immunoprecipitation assays. PGE(2)-induced internalization of PS-1 and co-localization of EP(4), PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP(4) receptor null mice. These results suggest that PGE(2)-stimulated production of Abeta involves EP(4) receptor-mediated endocytosis of PS-1 followed by activation of the gamma-secretase, as well as EP(2) receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.

Alcadein cleavages by amyloid beta-precursor protein (APP) alpha- and gamma-secretases generate small peptides, p3-Alcs, indicating Alzheimer disease-related gamma-secretase dysfunction.

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc(alpha), Alc(beta), and Alc(gamma). The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP alpha-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent gamma-secretase complex, thereby generating "APP p3-like" and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma), whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor beta-amyloid species Abeta42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma) were not equivalent, suggesting that one type of gamma-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect gamma-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.

Amyloid-dependent triosephosphate isomerase nitrotyrosination induces glycation and tau fibrillation.

Alzheimer's disease neuropathology is characterized by neuronal death, amyloid beta-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimer's disease, the molecular mechanisms linking amyloid beta-peptide and paired helical filaments remain unknown. Here, we show that amyloid beta-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimer's disease patients. Higher levels of nitro-triosephosphate isomerase (P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimer's disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal (n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimer's disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large beta-sheet aggregates in vitro and in vivo, as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimer's disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimer's disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid beta-peptide-induced toxicity and tau pathology.

Dissection of FUS domains involved in regulation of SnRNP70 gene expression.

FUS is one of the causative factors of amyotrophic lateral sclerosis. Loss and/or gain of its physiological functions has been believed to be linked to the pathogenesis of this condition. However, its functions remain incompletely understood. This study dissected the domains of FUS regulating the expression of SnRNP70, which functions in mRNA splicing. Biochemical analysis revealed that all FUS domains except for RGG1 contribute to determining Snrnp70 transcript abundance and thus its protein abundance. RNA-Seq analysis using the Gly-rich domain-deleted mutant coupled with snRNP70 knockdown revealed that FUS has a potential to regulate gene expression in both snRNP70-dependent and snRNP70-independent manners through the Gly-rich domain. These results provide insight into molecular details of the regulation of gene expression by FUS.

Suppression of APP-containing vesicle trafficking and production of beta-amyloid by AID/DHHC-12 protein.

The metabolism of amyloid beta-protein precursor (APP) is regulated by various cytoplasmic and/or membrane-associated proteins, some of which are involved in the regulation of intracellular membrane trafficking. We found that a protein containing Asp-His-His-Cys (DHHC) domain, alcadein and APP interacting DHHC protein (AID)/DHHC-12, strongly inhibited APP metabolism, including amyloid beta-protein (Abeta) generation. In cells expressing AID/DHHC-12, APP was tethered in the Golgi, and APP-containing vesicles disappeared from the cytoplasm. Although DHHC domain-containing proteins are involved in protein palmitoylation, a AID/DHHC-12 mutant of which the enzyme activity was impaired by replacing the DHHC sequence with Ala-Ala-His-Ser (AAHS) made no detectable difference in the generation and trafficking of APP-containing vesicles in the cytoplasm or the metabolism of APP. Furthermore, the mutant AID/DHHC-12 significantly increased non-amyloidogenic alpha-cleavage of APP along with activation of a disintegrin and metalloproteinase 17, a major alpha-secretase, suggesting that protein palmitoylation involved in the regulation of alpha-secretase activity. AID/DHHC-12 can modify APP metabolism, including Abeta generation in multiple ways by regulating the generation and/or trafficking of APP-containing vesicles from the Golgi and their entry into the late secretary pathway in an enzymatic activity-independent manner, and the alpha-cleavage of APP in the enzymatic activity-dependent manner.

Phosphorylation of the amino-terminal region of X11L regulates its interaction with APP.

X11-like (X11L) is neuronal adaptor protein that interacts with the amyloid beta-protein precursor (APP) and regulates its metabolism. The phosphotyrosine interaction/binding (PI/PTB) domain of X11L interacts with the cytoplasmic region of APP695. We found that X11L-APP interaction is enhanced in osmotically stressed cells and X11L modification is required for the enhancement. Amino acids 221-250 (X11L(221-250)) are required for the enhanced association with APP in osmotically stressed cells; this motif is 118 amino acids closer to the amino-terminal end of the protein than the PI/PTB domain (amino acids 368-555). We identified two phosphorylatable seryl residues, Ser236 and Ser238, in X11L(221-250) and alanyl substitution of either seryl residue diminished the enhanced association with APP. In brain Ser238 was found to be phosphorylated and phosphorylation of X11L was required for the interaction of X11L and APP. Both seryl residues in X11L(221-250) are conserved in neuronal X11, but not in X11L2, a non-neuronal X11 family member that did not exhibit enhanced APP association in osmotically stressed cells. These findings indicate that the region of X11L that regulates association with APP is located outside of, and amino-terminal to, the PI/PTB domain. Modification of this regulatory region may alter the conformation of the PI/PTB domain to modulate APP binding.

Novel Notch signaling inhibitor NSI­1 suppresses nuclear translocation of the Notch intracellular domain.

The Notch receptor serves a fundamental role in the regulation of cell fate determination through intracellular signal transmission. Mutation of the Notch receptor results in abnormal active signaling, leading to the development of diseases involving abnormal cell growth, including malignant tumors. Therefore, the Notch signaling pathway is a useful pharmacological target for the treatment of cancer. In the present study, a compound screening system was designed to identify inhibitors of the Notch signaling targeting Notch intracellular domain (NICD). A total of 9,600 compounds were analyzed using the Michigan Cancer Foundation­7 (MCF7) human breast adenocarcinoma cell line and the SH­SY5Y human neuroblastoma cell line with the reporter assay system using an artificial protein encoding a partial Notch carboxyl­terminal fragment fused to the Gal4 DNA­binding domain. The molecular mechanism underlying the inhibition of Notch signaling by a hit compound was further validated using biochemical and cell biological approaches. Using the screening system, a potential candidate, Notch signaling inhibitor­1 (NSI­1), was isolated which showed 50% inhibition at 6.1 µM in an exogenous Notch signaling system. In addition, NSI­1 suppressed the nuclear translocation of NICD and endogenous gene expression of hairy and enhancer of split­1, indicating that NSI­1 specifically targets Notch. Notably, NSI­1 suppressed the cell viability of MCF7 cells and another human breast adenocarcinoma cell line, MDA­MB­231 exhibiting constitutive and high Notch signaling activity, whereas no significant effect was observed in the SH­SY5Y cells bearing a lower Notch signaling activity. NSI­1 significantly suppressed the viability of SH­SY5Y cells expressing exogenous human Notch1. These results indicate that NSI­1 is a novel Notch signaling inhibitor and suggest its potential as a useful drug for the treatment of diseases induced by constitutively active Notch signaling.