TOLLIP acts as a cargo adaptor to promote lysosomal degradation of aberrant ER membrane proteins.

Endoplasmic reticulum (ER) proteostasis is maintained by various catabolic pathways. Lysosomes clear entire ER portions by ER-phagy, while proteasomes selectively clear misfolded or surplus aberrant proteins by ER-associated degradation (ERAD). Recently, lysosomes have also been implicated in the selective clearance of aberrant ER proteins, but the molecular basis remains unclear. Here, we show that the phosphatidylinositol-3-phosphate (PI3P)-binding protein TOLLIP promotes selective lysosomal degradation of aberrant membrane proteins, including an artificial substrate and motoneuron disease-causing mutants of VAPB and Seipin. These cargos are recognized by TOLLIP through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain. In contrast to ER-phagy receptors, which clear both native and aberrant proteins by ER-phagy, TOLLIP selectively clears aberrant cargos by coupling them with the PI3P-dependent lysosomal trafficking without promoting bulk ER turnover. Moreover, TOLLIP depletion augments ER stress after ERAD inhibition, indicating that TOLLIP and ERAD cooperatively safeguard ER proteostasis. Our study identifies TOLLIP as a unique type of cargo-specific adaptor dedicated to the clearance of aberrant ER cargos and provides insights into molecular mechanisms underlying lysosome-mediated quality control of membrane proteins.

Structures of α-synuclein filaments from multiple system atrophy.

Synucleinopathies, which include multiple system atrophy (MSA), Parkinson's disease, Parkinson's disease with dementia and dementia with Lewy bodies (DLB), are human neurodegenerative diseases1. Existing treatments are at best symptomatic. These diseases are characterized by the presence of, and believed to be caused by the formation of, filamentous inclusions of α-synuclein in brain cells2,3. However, the structures of α-synuclein filaments from the human brain are unknown. Here, using cryo-electron microscopy, we show that α-synuclein inclusions from the brains of individuals with MSA are made of two types of filament, each of which consists of two different protofilaments. In each type of filament, non-proteinaceous molecules are present at the interface of the two protofilaments. Using two-dimensional class averaging, we show that α-synuclein filaments from the brains of individuals with MSA differ from those of individuals with DLB, which suggests that distinct conformers or strains characterize specific synucleinopathies. As is the case with tau assemblies4-9, the structures of α-synuclein filaments extracted from the brains of individuals with MSA differ from those formed in vitro using recombinant proteins, which has implications for understanding the mechanisms of aggregate propagation and neurodegeneration in the human brain. These findings have diagnostic and potential therapeutic relevance, especially because of the unmet clinical need to be able to image filamentous α-synuclein inclusions in the human brain.

Presenilin deficiency enhances tau phosphorylation and its secretion.

Alzheimer's disease (AD) is characterized by the accumulation of abnormally folded amyloid ß-protein (Aß) in the brain parenchyma and phosphorylated tau in neurons. Presenilin (PS, PSEN) 1 and PS2 are essential components of γ-secretase, which is responsible for the cleavage of amyloid precursor protein (APP) to generate Aß. PSEN mutations are associated with tau aggregation in frontotemporal dementia, regardless of the presence or absence of Aß pathology. However, the mechanism by which PS regulates tau aggregation is still unknown. Here, we found that tau phosphorylation and secretion were significantly increased in PS double-knock-out (PS1/2-/-) fibroblasts compared with wild-type fibroblasts. Tau-positive vesicles in the cytoplasm were significantly increased in PS1/2-/- fibroblasts. Active GSK-3ß was increased in PS1/2-/- fibroblasts, and inhibiting GSK3ß activity in PS1/2-/- fibroblasts resulted in decreased tau phosphorylation and secretion. Transfection of WT human PS1 and PS2 reduced the secretion of phosphorylated tau and active GSK-3ß in PS1/2-/- fibroblasts. However, PS1D257A without γ-secretase activity did not decrease the secretion of phosphorylated tau. Furthermore, nicastrin deficiency also increased tau phosphorylation and secretion. These results suggest that deficient PS complex maturation may increase tau phosphorylation and secretion. Thus, our studies discover a new pathway by which PS regulates tau phosphorylation/secretion and pathology independent of Aß and suggest that PS serves as a potential therapeutic target for treating neurodegenerative diseases involving tau aggregation.

Effects of bound nucleotides on the secondary structure, thermal stability, and phosphorylation of Rab3A.

Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat kinase 2 (LRRK2) that is implicated in both familial and sporadic forms of Parkinson's disease (PD), and an abnormal increase in Rab3A phosphorylation has been proposed as a cause of PD. Despite the potential importance of Rab3A in PD pathogenesis, its structural information is limited and the effects of bound nucleotides on its biophysical and biochemical properties remain unclear. Here, we show that GDP-bound Rab3A is preferentially phosphorylated by LRRK2 compared with GTP-bound Rab3A. The secondary structure of Rab3A, measured by circular dichroism (CD) spectroscopy, revealed that Rab3A is resistant to heat-induced denaturation at pH 7.4 or 9.0 regardless of the nucleotides bound. In contrast, Rab3A underwent heat-induced denaturation at pH 5.0 at a lower temperature in its GDP-bound form than in its GTP-bound form. The unfolding temperature of Rab3A was studied by differential scanning fluorimetry, which showed a significantly higher unfolding temperature in GTP-bound Rab3A than in GDP-bound Rab3A, with the highest at pH 7.4. These results suggest that Rab3A has unusual thermal stability under physiologically relevant conditions and that bound nucleotides influence both thermal stability and phosphorylation by LRRK2.

Pathogenic LRRK2 compromises the subcellular distribution of lysosomes in a Rab12-RILPL1-dependent manner.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). Recent studies have shown that LRRK2 physiologically phosphorylates several Rab family proteins including Rab12 and that this phosphorylation is accelerated by the pathogenic mutations in LRRK2, although the significance in the PD pathogenesis remains unknown. Here we examined the effect of the overexpression of LRRK2 on the distribution of organelles in cultured cells and found that lysosomes become clustered in a perinuclear region upon the overexpression of pathogenic mutant LRRK2 in a manner dependent on its kinase activity. The perinuclear clustering of lysosomes was abolished by knocking out RAB12 as well as its effector protein RILPL1. Re-expression of Rab12 in RAB12 knockout cells suggested that the phosphorylation at Ser106 of Rab12 is required for the perinuclear clustering of lysosomes. Moreover, phosphorylated Rab12 was also accumulated on the clustered lysosomes, and the phosphorylation of Rab12 increased its interaction with RILPL1, leading us to conclude that the increase in the phosphorylation of Rab12 by pathogenic LRRK2 compromised intracellular lysosomal transport via the enhanced interaction of Rab12 with RILPL1. These data suggest the involvement of abnormal regulation of lysosomal transport in the LRRK2-mediated pathogenesis of PD.

Photo-oxygenation of histidine residue inhibits α-synuclein aggregation.

Aggregation of α-synuclein (α-syn) into amyloid is the pathological hallmark of several neurodegenerative disorders, including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. It is widely accepted that α-syn aggregation is associated with neurodegeneration, although the mechanisms are not yet fully understood. Therefore, the inhibition of α-syn aggregation is a potential therapeutic approach against these diseases. This study used the photocatalyst for α-syn photo-oxygenation, which selectively adds oxygen atoms to fibrils. Our findings demonstrate that photo-oxygenation using this photocatalyst successfully inhibits α-syn aggregation, particularly by reducing its seeding ability. Notably, we also discovered that photo-oxygenation of the histidine at the 50th residue in α-syn aggregates is responsible for the inhibitory effect. These findings indicate that photo-oxygenation of the histidine residue in α-syn is a potential therapeutic strategy for synucleinopathies.

ADAMTS4 is involved in the production of the Alzheimer disease amyloid biomarker APP669-711.

Amyloid-ß (Aß) deposition in the brain parenchyma is one of the pathological hallmarks of Alzheimer disease (AD). We have previously identified amyloid precursor protein (APP)669-711 (a.k.a. Aß(-3)-40) in human plasma using immunoprecipitation combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IP-MALDI-MS). Furthermore, we found that the level of a composite biomarker, i.e., a combination of APP669-711/Aß1-42 ratio and Aß1-40/Aß1-42 ratio in human plasma, correlates with the amyloid PET status of AD patients. However, the production mechanism of APP669-711 has remained unclear. Using in vitro and in vivo assays, we identified A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) as a responsible enzyme for APP669-711 production. ADAMTS4 cleaves APP directly to generate the C-terminal stub c102, which is subsequently proteolyzed by γ-secretase to release APP669-711. Genetic knockout of ADAMTS4 reduced the production of endogenous APP669-711 by 30% to 40% in cultured cells as well as mouse plasma, irrespectively of Aß levels. Finally, we found that the endogenous murine APP669-711/Aß1-42 ratio was increased in aged AD model mice, which shows Aß deposition as observed in human patients. These data suggest that ADAMTS4 is involved in the production of APP669-711, and a plasma biomarker determined by IP-MALDI-MS can be used to estimate the level of Aß deposition in the brain of mouse models.

Distinct tau folds initiate templated seeding and alter the post-translational modification profile.

Pathological tau accumulates in the brain in tauopathies such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy and corticobasal degeneration, and forms amyloid-like filaments incorporating various post-translational modifications (PTMs). Cryo-electron microscopic (cryo-EM) studies have demonstrated that tau filaments extracted from tauopathy brains are characteristic of the disease and share a common fold(s) in the same disease group. Furthermore, the tau PTM profile changes during tau pathology formation and disease progression, and disease-specific PTMs are detected in and around the filament core. In addition, templated seeding has been suggested to trigger pathological tau amplification and spreading in vitro and in vivo, although the molecular mechanisms are not fully understood. Recently, we reported that the cryo-EM structures of tau protofilaments in SH-SY5Y cells seeded with patient-derived tau filaments show a core structure(s) resembling that of the original seeds. Here, we investigated PTMs of tau filaments accumulated in the seeded cells by liquid chromatography/tandem mass spectrometry and compared them with the PTMs of patient-derived tau filaments. Examination of insoluble tau extracted from SH-SY5Y cells showed that numerous phosphorylation, deamidation and oxidation sites detected in the fuzzy coat in the original seeds were well reproduced in SH-SY5Y cells. Moreover, templated tau filament formation preceded both truncation of the N-/C-terminals of tau and PTMs in and around the filament core, indicating these PTMs may predominantly be introduced after the degradation of the fuzzy coat.

High performance plasma amyloid-ß biomarkers for Alzheimer's disease.

To facilitate clinical trials of disease-modifying therapies for Alzheimer's disease, which are expected to be most efficacious at the earliest and mildest stages of the disease, supportive biomarker information is necessary. The only validated methods for identifying amyloid-ß deposition in the brain-the earliest pathological signature of Alzheimer's disease-are amyloid-ß positron-emission tomography (PET) imaging or measurement of amyloid-ß in cerebrospinal fluid. Therefore, a minimally invasive, cost-effective blood-based biomarker is desirable. Despite much effort, to our knowledge, no study has validated the clinical utility of blood-based amyloid-ß markers. Here we demonstrate the measurement of high-performance plasma amyloid-ß biomarkers by immunoprecipitation coupled with mass spectrometry. The ability of amyloid-ß precursor protein (APP)669-711/amyloid-ß (Aß)1-42 and Aß1-40/Aß1-42 ratios, and their composites, to predict individual brain amyloid-ß-positive or -negative status was determined by amyloid-ß-PET imaging and tested using two independent data sets: a discovery data set (Japan, n = 121) and a validation data set (Australia, n = 252 including 111 individuals diagnosed using 11C-labelled Pittsburgh compound-B (PIB)-PET and 141 using other ligands). Both data sets included cognitively normal individuals, individuals with mild cognitive impairment and individuals with Alzheimer's disease. All test biomarkers showed high performance when predicting brain amyloid-ß burden. In particular, the composite biomarker showed very high areas under the receiver operating characteristic curves (AUCs) in both data sets (discovery, 96.7%, n = 121 and validation, 94.1%, n = 111) with an accuracy approximately equal to 90% when using PIB-PET as a standard of truth. Furthermore, test biomarkers were correlated with amyloid-ß-PET burden and levels of Aß1-42 in cerebrospinal fluid. These results demonstrate the potential clinical utility of plasma biomarkers in predicting brain amyloid-ß burden at an individual level. These plasma biomarkers also have cost-benefit and scalability advantages over current techniques, potentially enabling broader clinical access and efficient population screening.

Presenilin Is Essential for ApoE Secretion, a Novel Role of Presenilin Involved in Alzheimer's Disease Pathogenesis.

Alzheimer's disease (AD) is a debilitating dementia characterized by progressive memory loss and aggregation of amyloid-ß (Aß) protein into amyloid plaques in patient brains. Mutations in presenilin (PS) lead to abnormal generation of Aß, which is the major cause of familial AD (FAD), and apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) onset. However, whether dysfunction of PS is involved in the pathogenesis of SAD is largely unknown. We found that ApoE secretion was completely abolished in PS-deficient cells and markedly decreased by inhibition of γ-secretase activity. Blockade of γ-secretase activity by a γ-secretase inhibitor, DAPT, decreased ApoE secretion, suggesting an important role of γ-secretase activity in ApoE secretion. Reduced ApoE secretion is also observed in nicastrin-deficient cells with reduced γ-secretase activity. PS deficiency enhanced nuclear translocation of ApoE and binding of ApoE to importin α4, a nuclear transport receptor. Moreover, the expression of PS mutants in PS-deficient cells suppressed the restoration effects on ApoE secretion compared with the expression of wild-type PS. Plasma ApoE levels were lower in FAD patients carrying PS1 mutations compared with normal control subjects. Our findings suggest a novel role of PS contributing to the pathogenesis of SAD by regulating ApoE secretion.SIGNIFICANCE STATEMENT Familial AD (FAD) typically results from mutations in the genes encoding amyloid precursor protein, presenilin 1 (PS1), or PS2. Many PS mutants have been found to exert impaired γ-secretase activity and increased amyloid-ß 42 (Aß42)/Aß40 ratio, which induce early amyloid deposition and FAD. On the other hand, apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) and contributes to AD pathogenesis because it has reduced Aß clearance capability compared with ApoE3 and ApoE2. FAD and SAD have long been considered to be caused by these two independent mechanisms; however, for the first time, we demonstrated that PS is essential for ApoE secretion and PS mutants affected ApoE secretion in vitro and in human samples, suggesting a novel mechanism by which PS is also involved in SAD pathogenesis.

The atypical Rab GTPase associated with Parkinson's disease, Rab29, is localized to membranes.

Several genetic studies have shown that the small GTPase Rab29 is involved in the pathogenesis of Parkinson's Disease (PD). It has also been shown that overexpression of Rab29 increases the activity of leucine-rich repeat kinase 2, a protein kinase often mutated in familial PD, although the mechanism underlying this activation remains unclear. Here, we employed biochemical analyses to characterize the localization of Rab29 and found that, unlike general Rab proteins, Rab29 is predominantly fractionated into the membrane fraction by ultracentrifugation. We also found that Rab29 is resistant to extraction from membranes by GDP-dissociation inhibitors (GDIs) in vitro. Furthermore, Rab29 failed to interact with GDIs, and its membrane localization was not affected by the knockout of GDIs in cells. We show that the knockout of Rab geranylgeranyltransferase decreased the hydrophobicity of Rab29, suggesting that Rab29 is geranylgeranylated at its carboxyl terminus as is with typical Rab proteins. Notably, we demonstrated that membrane-bound Rab29 retains some hydrophilicity, indicating that mechanisms other than geranylgeranylation might also be involved in the membrane localization of Rab29. Taken together, these findings uncover the atypical nature of Rab29 among Rab proteins, which will provide important clues for understanding how Rab29 is involved in the molecular pathomechanism of PD.

BORCS6 is involved in the enlargement of lung lamellar bodies in Lrrk2 knockout mice.

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson disease. It has been shown that Lrrk2 knockout (KO) rodents have enlarged lamellar bodies (LBs) in their alveolar epithelial type II cells, although the underlying mechanisms remain unclear. Here we performed proteomic analyses on LBs isolated from Lrrk2 KO mice and found that the LB proteome is substantially different in Lrrk2 KO mice compared with wild-type mice. In Lrrk2 KO LBs, several Rab proteins were increased, and subunit proteins of BLOC-1-related complex (BORC) were decreased. The amount of surfactant protein C was significantly decreased in the bronchoalveolar lavage fluid obtained from Lrrk2 KO mice, suggesting that LB exocytosis is impaired in Lrrk2 KO mice. We also found that the enlargement of LBs is recapitulated in A549 cells upon KO of LRRK2 or by treating cells with LRRK2 inhibitors. Using this model, we show that KO of BORCS6, a BORC subunit gene, but not other BORC genes, causes LB enlargement. Our findings implicate the LRRK2-BORCS6 pathway in the maintenance of LB morphology.

LRRK2-mediated phosphorylation and thermal stability of Rab12 are regulated by bound nucleotides.

An abnormal increase in the phosphorylation of Rab12 by leucine-rich repeat kinase 2 (LRRK2), a serine/threonine kinase genetically linked to Parkinson's disease (PD), has been implicated in the pathogenesis of PD, although the underlying mechanism remains unclear. In this report, we show that LRRK2 phosphorylates Rab12 more efficiently in its GDP-bound form than in its GTP-bound form using an in vitro phosphorylation assay. This observation suggests that LRRK2 recognizes the structural difference of Rab12 caused by the bound nucleotide and that Rab12 phosphorylation inhibits its activation. Circular dichroism data revealed that Rab12, in its GDP-bound form, is more susceptible to heat-induced denaturation than its GTP-bound form, which was exacerbated at basic pH. Differential scanning fluorimetry showed that heat-induced denaturation of Rab12 in its GDP-bound form occurs at a lower temperature than in its GTP-bound form. These results suggest that the type of nucleotide bound to Rab12 determines the efficiency of LRRK2-mediated phosphorylation and the thermal stability of Rab12, and provide insights into elucidating the mechanism of the abnormal increase in Rab12 phosphorylation.

Pathological Roles of INPP5D in Alzheimer's Disease.

Current hypothesis of Alzheimer's disease (AD) postulates that amyloid ß (Aß) deposition in the brain causes tau inclusion in neurons and leads to cognitive decline. The discovery of the genetic association between triggering receptor expressed on myeloid cells 2 (TREM2) with increased AD risk points to a causal link between microglia and AD pathogenesis, and revealed a crucial role of TREM2-dependent clustering of microglia around amyloid plaques that prevents Aß toxicity to facilitate tau deposition near the plaques. Here we review the physiological and pathological roles of another AD risk gene expressed in microglia, inositol polyphosphate-5-polyphosphatase D (INPP5D), which encodes a phosphoinositide phosphatase. Evidence suggests that its risk polymorphisms alter the expression level and/or function of INPP5D, while concomitantly affecting tau levels in cerebrospinal fluids. In ß-amyloidosis mice, INPP5D was upregulated upon Aß deposition and negatively regulated the microglial clustering toward amyloid plaques. INPP5D seems to exert its function by acting antagonistically at downstream of the TREM2 signaling pathway, suggesting that it is a novel regulator of the protective barrier by microglia. Further studies to elucidate INPP5D's role in AD may help in developing new therapeutic targets for AD treatment.

GPR120 signaling controls amyloid-ß degrading activity of matrix metalloproteinases.

Alzheimer disease (AD) is characterized by the extensive deposition of amyloid-ß peptide (Aß) in the brain. Brain Aß level is regulated by a balance between Aß production and clearance. The clearance rate of Aß is decreased in the brains of sporadic AD patients, indicating that the dysregulation of Aß clearance mechanisms affects the pathological process of AD. Astrocytes are among the most abundant cells in the brain and are implicated in the clearance of brain Aß via their regulation of the blood-brain barrier, glymphatic system, and proteolytic degradation. The cellular morphology and activity of astrocytes are modulated by several molecules, including ω3 polyunsaturated fatty acids, such as docosahexaenoic acid, which is one of the most abundant lipids in the brain, via the G protein-coupled receptor GPR120/FFAR4. In this study, we analyzed the role of GPR120 signaling in the Aß-degrading activity of astrocytes. Treatment with the selective antagonist upregulated the matrix metalloproteinase (MMP) inhibitor-sensitive Aß-degrading activity in primary astrocytes. Moreover, the inhibition of GPR120 signaling increased the levels of Mmp2 and Mmp14 mRNAs, and decreased the expression levels of tissue inhibitor of metalloproteinases 3 (Timp3) and Timp4, suggesting that GPR120 negatively regulates the astrocyte-derived MMP network. Finally, the intracerebral injection of GPR120 specific antagonist substantially decreased the levels of Tris-buffered saline-soluble Aß in male AD model mice, and this effect was canceled by the coinjection of an MMP inhibitor. These data indicate that astrocytic GPR120 signaling negatively regulates the Aß degrading activity of MMPs.SIGNIFICANT STATEMENTThe level of amyloid ß (Aß) in the brain is a crucial determinant of the development of Alzheimer disease. Here we found that astrocytes, which are the most abundant cell type in the central nervous system, harbors degrading activity against amyloid ß, which is regulated by GPR120 signaling. GPR120 is involved in the inflammatory response and obesity in peripheral organs. However, the pathophysiological role of GPR120 in Alzheimer disease remains unknown. We found that selective inhibition of GPR120 signaling in astrocytes increased the Aß-degrading activity of matrix metalloproteases. Our results suggest that GPR120 in astrocytes is a novel therapeutic target for the development of anti-Aß therapeutics.

Structure-activity relationship of presenilin in γ-secretase-mediated intramembrane cleavage.

Genetic research on familial cases of Alzheimer disease have identified presenilin (PS) as an important membrane protein in the pathomechanism of this disease. PS is the catalytic subunit of γ-secretase, which is responsible for the generation of amyloid-ß peptide deposited in the brains of Alzheimer disease patients. γ-Secretase is an atypical protease composed of four membrane proteins (i.e., presenilin, nicastrin, anterior pharynx defective-1 (Aph-1), and presenilin enhancer-2 (Pen-2)) and mediates intramembrane proteolysis. Numerous investigations have been conducted toward understanding the structural features of γ-secretase components as well as the cleavage mechanism of γ-secretase. In this review, we summarize our current understanding of the structure and activity relationship of the γ-secretase complex.

Photo-oxygenation by a biocompatible catalyst reduces amyloid-ß levels in Alzheimer's disease mice.

Amyloid formation and the deposition of the amyloid-ß peptide are hallmarks of Alzheimer's disease pathogenesis. Immunotherapies using anti-amyloid-ß antibodies have been highlighted as a promising approach for the prevention and treatment of Alzheimer's disease by enhancing microglial clearance of amyloid-ß peptide. However, the efficiency of antibody delivery into the brain is limited, and therefore an alternative strategy to facilitate the clearance of brain amyloid is needed. We previously developed an artificial photo-oxygenation system using a low molecular weight catalytic compound. The photocatalyst specifically attached oxygen atoms to amyloids upon irradiation with light, and successfully reduced the neurotoxicity of aggregated amyloid-ß via inhibition of amyloid formation. However, the therapeutic effect and mode of actions of the photo-oxygenation system in vivo remained unclear. In this study, we demonstrate that photo-oxygenation facilitates the clearance of aggregated amyloid-ß from the brains of living Alzheimer's disease model mice, and enhances the microglial degradation of amyloid-ß peptide. These results suggest that photo-oxygenation may represent a novel anti-amyloid-ß strategy in Alzheimer's disease, which is compatible with immunotherapy.

Human tauopathy-derived tau strains determine the substrates recruited for templated amplification.

Tauopathies are a subset of neurodegenerative diseases characterized by abnormal tau inclusions. Specifically, three-repeat tau and four-repeat tau in Alzheimer's disease, three-repeat tau in Pick's disease (PiD) and four-repeat tau in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) form amyloid-like fibrous structures that accumulate in neurons and/or glial cells. Amplification and cell-to-cell transmission of abnormal tau based on the prion hypothesis are believed to explain the onset and progression of tauopathies. Recent studies support not only the self-propagation of abnormal tau, but also the presence of conformationally distinct tau aggregates, namely tau strains. Cryogenic electron microscopy analyses of patient-derived tau filaments have revealed disease-specific ordered tau structures. However, it remains unclear whether the ultrastructural and biochemical properties of tau strains are inherited during the amplification of abnormal tau in the brain. In this study, we investigated template-dependent amplification of tau aggregates using a cellular model of seeded aggregation. Tau strains extracted from human tauopathies caused strain-dependent accumulation of insoluble filamentous tau in SH-SY5Y cells. The seeding activity towards full-length four-repeat tau substrate was highest in CBD-tau seeds, followed by PSP-tau and Alzheimer's disease (AD)-tau seeds, while AD-tau seeds showed higher seeding activity than PiD-tau seeds towards three-repeat tau substrate. Abnormal tau amplified in cells inherited the ultrastructural and biochemical properties of the original seeds. These results strongly suggest that the structural differences of patient-derived tau strains underlie the diversity of tauopathies, and that seeded aggregation and filament formation mimicking the pathogenesis of sporadic tauopathy can be reproduced in cultured cells. Our results indicate that the disease-specific conformation of tau aggregates determines the tau isoform substrate that is recruited for templated amplification, and also influences the prion-like seeding activity.

Specific Mutations in Aph1 Cause γ-Secretase Activation.

Amyloid beta peptides (Aßs) are generated from amyloid precursor protein (APP) through multiple cleavage steps mediated by γ-secretase, including endoproteolysis and carboxypeptidase-like trimming. The generation of neurotoxic Aß42/43 species is enhanced by familial Alzheimer's disease (FAD) mutations within the catalytic subunit of γ-secretase, presenilin 1 (PS1). FAD mutations of PS1 cause partial loss-of-function and decrease the cleavage activity. Activating mutations, which have the opposite effect of FAD mutations, are important for studying Aß production. Aph1 is a regulatory subunit of γ-secretase; it is presumed to function as a scaffold of the complex. In this study, we identified Aph1 mutations that are active in the absence of nicastrin (NCT) using a yeast γ-secretase assay. We analyzed these Aph1 mutations in the presence of NCT; we found that the L30F/T164A mutation is activating. When introduced in mouse embryonic fibroblasts, the mutation enhanced cleavage. The Aph1 mutants produced more short and long Aßs than did the wild-type Aph1, without an apparent modulatory function. The mutants did not change the amount of γ-secretase complex, suggesting that L30F/T164A enhances catalytic activity. Our results provide insights into the regulatory function of Aph1 in γ-secretase activity.

Suppression of amyloid-ß secretion from neurons by cis-9, trans-11-octadecadienoic acid, an isomer of conjugated linoleic acid.

Two common conjugated linoleic acids (LAs), cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA), exert various biological activities. However, the effect of CLA on the generation of neurotoxic amyloid-ß (Aß) protein remains unclear. We found that c9,t11 CLA significantly suppressed the generation of Aß in mouse neurons. CLA treatment did not affect the level of ß-site APP-cleaving enzyme 1 (BACE1), a component of active γ-secretase complex presenilin 1 amino-terminal fragment, or Aß protein precursor (APP) in cultured neurons. BACE1 and γ-secretase activities were not directly affected by c9,t11 CLA. Localization of BACE1 and APP in early endosomes increased in neurons treated with c9,t11 CLA; concomitantly, the localization of both proteins was reduced in late endosomes, the predominant site of APP cleavage by BACE1. The level of CLA-containing phosphatidylcholine (CLA-PC) increased dramatically in neurons incubated with CLA. Incorporation of phospholipids containing c9,t11 CLA, but not t10,c12 CLA, into the membrane may affect the localization of some membrane-associated proteins in intracellular membrane compartments. Thus, in neurons treated with c9,t11 CLA, reduced colocalization of APP with BACE1 in late endosomes may decrease APP cleavage by BACE1 and subsequent Aß generation. Our findings suggest that the accumulation of c9,t11 CLA-PC/LPC in neuronal membranes suppresses the production of neurotoxic Aß in neurons.