Short-term regulation of TSFM level does not alter amyloidogenesis and mitochondrial function in type-specific cells.

BACKGROUND: Mitochondrial Ts translation elongation factor (TSFM) is an enzyme that catalyzes exchange of guanine nucleotides. By forming a complex with mitochondrial Tu translation elongation factor (TUFM), TSFM participates in mitochondrial protein translation. We have previously reported that TUFM regulates translation of beta-site APP cleaving enzyme 1 (BACE1) via ROS (reactive oxygen species)-dependent mechanism, suggesting a potential role in amyloid precursor protein (APP) processing associated with Alzheimer's disease (AD), which led to the speculation that TSFM may regulate APP processing in a similar way to TUFM. METHODS AND RESULTS: Here, we report that in cultured cells, knockdown or overexpression TSFM did not change protein levels in BACE1 and APP. Besides, the levels of cytoplasmic ROS and mitochondrial superoxide, in addition to ATP level, cell viability and mitochondrial membrane potential were not significantly altered by TSFM knockdown in the short term. Further transcriptome analysis revealed that expression of majority of mitochondrial genes were not remarkably changed by TSFM silencing. The possibility of TSFM involved in cardiomyopathy and cancer development was uncovered using bioinformatics analysis. CONCLUSIONS: Collectively, short-term regulation of TSFM level in cultured cells does not cause a significant change in proteins involved in APP processing, levels in ROS and ATP associated with mitochondrial function. Whereas our study could contribute to comprehend certain clinical features of TSFM mutations, the roles of TSFM in cardiomyopathy and cancer development might deserve further investigation.

Simple modeling of familial Alzheimer's disease using human pluripotent stem cell-derived cerebral organoid technology.

BACKGROUND: Cerebral organoids (COs) are the most advanced in vitro models that resemble the human brain. The use of COs as a model for Alzheimer's disease (AD), as well as other brain diseases, has recently gained attention. This study aimed to develop a human AD CO model using normal human pluripotent stem cells (hPSCs) that recapitulates the pathological phenotypes of AD and to determine the usefulness of this model for drug screening. METHODS: We established AD hPSC lines from normal hPSCs by introducing genes that harbor familial AD mutations, and the COs were generated using these hPSC lines. The pathological features of AD, including extensive amyloid-ß (Aß) accumulation, tauopathy, and neurodegeneration, were analyzed using enzyme-linked immunosorbent assay, Amylo-Glo staining, thioflavin-S staining, immunohistochemistry, Bielschowsky's staining, and western blot analysis. RESULTS: The AD COs exhibited extensive Aß accumulation. The levels of paired helical filament tau and neurofibrillary tangle-like silver deposits were highly increased in the AD COs. The number of cells immunoreactive for cleaved caspase-3 was significantly increased in the AD COs. In addition, treatment of AD COs with BACE1 inhibitor IV, a ß-secretase inhibitor, and compound E, a γ-secretase inhibitor, significantly attenuated the AD pathological features. CONCLUSION: Our model effectively recapitulates AD pathology. Hence, it is a valuable platform for understanding the mechanisms underlying AD pathogenesis and can be used to test the efficacy of anti-AD drugs.

Spatial-Temporal Mapping Reveals the Golgi as the Major Processing Site for the Pathogenic Swedish APP Mutation: Familial APP Mutant Shifts the Major APP Processing Site.

Alzheimer's disease is associated with increased levels of amyloid beta (Aß) generated by sequential intracellular cleavage of amyloid precursor protein (APP) by membrane-bound secretases. However, the spatial and temporal APP cleavage events along the trafficking pathways are poorly defined. Here, we use the Retention Using Selective Hooks (RUSH) to compare in real time the anterograde trafficking and temporal cleavage events of wild-type APP (APPwt) with the pathogenic Swedish APP (APPswe) and the disease-protective Icelandic APP (APPice). The analyses revealed differences in the trafficking profiles and processing between APPwt and the APP familial mutations. While APPwt was predominantly processed by the ß-secretase, BACE1, following Golgi transport to the early endosomes, the transit of APPswe through the Golgi was prolonged and associated with enhanced amyloidogenic APP processing and Aß secretion. A 20°C block in cargo exit from the Golgi confirmed ß- and γ-secretase processing of APPswe in the Golgi. Inhibition of the ß-secretase, BACE1, restored APPswe anterograde trafficking profile to that of APPwt. APPice was transported rapidly through the Golgi to the early endosomes with low levels of Aß production. This study has revealed different intracellular locations for the preferential cleavage of APPwt and APPswe and Aß production, and the Golgi as the major processing site for APPswe, findings relevant to understand the molecular basis of Alzheimer's disease.

A novel mouse model for N-terminal truncated Aß2-x generation through meprin ß overexpression in astrocytes.

Neurotoxic amyloid-ß (Aß) peptides cause neurodegeneration in Alzheimer's disease (AD) patients' brains. They are released upon proteolytic processing of the amyloid precursor protein (APP) extracellularly at the ß-secretase site and intramembranously at the γ-secretase site. Several AD mouse models were developed to conduct respective research in vivo. Most of these classical models overexpress human APP with mutations driving AD-associated pathogenic APP processing. However, the resulting pattern of Aß species in the mouse brains differs from those observed in AD patients' brains. Particularly mutations proximal to the ß-secretase cleavage site (e.g., the so-called Swedish APP (APPswe) fostering Aß1-x formation) lead to artificial Aß production, as N-terminally truncated Aß peptides are hardly present in these mouse brains. Meprin ß is an alternative ß-secretase upregulated in brains of AD patients and capable of generating N-terminally truncated Aß2-x peptides. Therefore, we aimed to generate a mouse model for the production of so far underestimated Aß2-x peptides by conditionally overexpressing meprin ß in astrocytes. We chose astrocytes as meprin ß was detected in this cell type in close proximity to Aß plaques in AD patients' brains. The meprin ß-overexpressing mice showed elevated amyloidogenic APP processing detected with a newly generated neo-epitope-specific antibody. Furthermore, we observed elevated Aß production from endogenous APP as well as AD-related behavior changes (hyperlocomotion and deficits in spatial memory). The novel mouse model as well as the established tools and methods will be helpful to further characterize APP cleavage and the impact of different Aß species in future studies.

Chicoric Acid Ameliorated Beta-Amyloid Pathology and Enhanced Expression of Synaptic-Function-Related Markers via L1CAM in Alzheimer's Disease Models.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disease. The accumulation of amyloid-beta (Aß) plaques is a distinctive pathological feature of AD patients. The aims of this study were to evaluate the therapeutic effect of chicoric acid (CA) on AD models and to explore its underlying mechanisms. APPswe/Ind SH-SY5Y cells and 5xFAD mice were treated with CA. Soluble Aß1-42 and Aß plaque levels were analyzed by ELISA and immunohistochemistry, respectively. Transcriptome sequencing was used to compare the changes in hippocampal gene expression profiles among the 5xFAD mouse groups. The specific gene expression levels were quantified by qRT-PCR and Western blot analysis. It was found that CA treatment reduced the Aß1-42 levels in the APPswe/Ind cells and 5xFAD mice. It also reduced the Aß plaque levels as well as the APP and BACE1 levels. Transcriptome analysis showed that CA affected the synaptic-plasticity-related genes in the 5xFAD mice. The levels of L1CAM, PSD-95 and synaptophysin were increased in the APPswe/Ind SH-SY5Y cells and 5xFAD mice treated with CA, which could be inhibited by administering siRNA-L1CAM to the CA-treated APPswe/Ind SH-SY5Y cells. In summary, CA reduced Aß levels and increased the expression levels of synaptic-function-related markers via L1CAM in AD models.

Alpha-, Beta-, and Gamma-Secretase, Amyloid Precursor Protein, and Tau Protein Genes in the Hippocampal CA3 Subfield in an Ischemic Model of Alzheimer's Disease with Survival up to 2 Years.

Background: Understanding the phenomena underlying the non-selective susceptibility to ischemia of pyramidal neurons in the CA3 is important from the point of view of elucidating the mechanisms of memory loss and the development of dementia. Objective: The aim of the study was to investigate changes in genes expression of amyloid precursor protein, its cleaving enzymes and tau protein in CA3 post-ischemia with survival of 12-24 months. Methods: We used an ischemic model of Alzheimer's disease to study the above genes using an RT-PCR protocol. Results: The expression of the amyloid precursor protein gene was above the control values at all times post-ischemia. The expression of the α-secretase gene also exceeded the control values post-ischemia. The expression of the ß-secretase gene increased 12 and 24 months post-ischemia, and 18 months was below control values. Presenilin 1 and 2 genes expression was significantly elevated at all times post-ischemia. Also, tau protein gene expression was significantly elevated throughout the observation period, and peak gene expression was present 12 months post-ischemia. Conclusions: The study suggests that the genes studied are involved in the non-amyloidogenic processing of amyloid precursor protein. Additionally data indicate that brain ischemia with long-term survival causes damage and death of pyramidal neurons in the CA3 area of the hippocampus in a modified tau protein-dependent manner. Thus defining a new and important mechanism of pyramidal neuronal death in the CA3 area post-ischemia. In addition expression of tau protein gene modification after brain ischemia is useful in identifying ischemic mechanisms occurring in Alzheimer's disease.

Targeting blood brain barrier-Remote ischemic conditioning alleviates cognitive impairment in female APP/PS1 rats.

AIMS: Alzheimer's disease (AD) is a significant global health concern, and it is crucial that we find effective methods to prevent or slow down AD progression. Recent studies have highlighted the essential role of blood vessels in clearing Aß, a protein that contributes to AD. Scientists are exploring blood biomarkers as a potential tool for future AD diagnosis. One promising method that may help prevent AD is remote ischemic conditioning (RIC). RIC involves using sub-lethal ischemic-reperfusion cycles on limbs. However, a comprehensive understanding of how RIC can prevent AD and its long-term effectiveness is still lacking. Further research is essential to fully comprehend the potential benefits of RIC in preventing AD. METHODS: Female wild-type (WT) and APP/PS1 transgenic rats, aged 12 months, underwent ovariectomy and were subsequently assigned to WT, APP/PS1, and APP/PS1 + RIC groups. RIC was conducted five times a week for 4 weeks. The rats' depressive and cognitive behaviors were evaluated using force swimming, open-field tests, novel objective recognition, elevated plus maze, and Barnes maze tests. Evaluation of the neurovascular unit (NVU), synapses, vasculature, astrocytes, and microglia was conducted using immunofluorescence staining (IF), Western blot (WB), and transmission electron microscopy (TEM). Additionally, the cerebro-vasculature was examined using micro-CT, and cerebral blood flow (CBF) was measured using Speckle Doppler. Blood-brain barrier (BBB) permeability was determined by measuring the Evans blue leakage. Finally, Aß levels in the rat frontal cortex were measured using WB, ELISA, or IF staining. RESULTS: RIC enhanced memory-related protein expression and rescued depressive-like behavior and cognitive decline in APP/PS1 transgenic rats. Additionally, the intervention protected NVU in the rat frontal cortex, as evidenced by (1) increased expression of TJ (tight junction) proteins, pericyte marker PDGFRß, and glucose transporter 1 (GLUT1), as well as decreased VCAM1; (2) mitigation of ultrastructure impairment in neuron, cerebral vascular, and astrocyte; (3) upregulation of A2 astrocyte phenotype markers and downregulation of A1 phenotype markers, indicating a shift toward a healthier phenotype. Correspondingly, RIC intervention alleviated neuroinflammation, as evidenced by the decreased Iba1 level, a microglia marker. Meanwhile, RIC intervention elevated CBF in frontal cortex of the rats. Notably, RIC intervention effectively suppressed Aß toxicity, as demonstrated by the enhancement of α-secretase and attenuation of ß-secretase (BACE1) and γ- secretase and Aß1-42 and Aß1-40 levels as well. CONCLUSION: Chronic RIC intervention exerts vascular and neuroprotective roles, suggesting that RIC could be a promising therapeutic strategy targeting the BBB and NVU during AD development.

New precision medicine avenues to the prevention of Alzheimer's disease from insights into the structure and function of γ-secretases.

Two phase-III clinical trials with anti-amyloid peptide antibodies have met their primary goal, i.e. slowing of Alzheimer's disease (AD) progression. However, antibody therapy may not be the optimal therapeutic modality for AD prevention, as we will discuss in the context of the earlier small molecules described as "γ-secretase modulators" (GSM). We review here the structure, function, and pathobiology of γ-secretases, with a focus on how mutations in presenilin genes result in early-onset AD. Significant progress has been made in generating compounds that act in a manner opposite to pathogenic presenilin mutations: they stabilize the proteinase-substrate complex, thereby increasing the processivity of substrate cleavage and altering the size spectrum of Aß peptides produced. We propose the term "γ-secretase allosteric stabilizers" (GSAS) to distinguish these compounds from the rather heterogenous class of GSM. The GSAS represent, in theory, a precision medicine approach to the prevention of amyloid deposition, as they specifically target a discrete aspect in a complex cell biological signalling mechanism that initiates the pathological processes leading to Alzheimer's disease.

Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes.

Mutations that cause familial Alzheimer's disease (FAD) are found in amyloid precursor protein (APP) and presenilin, the catalytic component of γ-secretase, that together produce amyloid ß-peptide (Aß). Nevertheless, whether Aß is the primary disease driver remains controversial. We report here that FAD mutations disrupt initial proteolytic events in the multistep processing of APP substrate C99 by γ-secretase. Cryoelectron microscopy reveals that a substrate mimetic traps γ-secretase during the transition state, and this structure aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and in cellulo fluorescence microscopy support stabilization of enzyme-substrate complexes by FAD mutations. Neuronal expression of C99 and/or presenilin-1 in Caenorhabditis elegans leads to synaptic loss only with FAD-mutant transgenes. Designed mutations that stabilize the enzyme-substrate complex and block Aß production likewise led to synaptic loss. Collectively, these findings implicate the stalled process-not the products-of γ-secretase cleavage of substrates in FAD pathogenesis.

The Large Ectodomain of APP Prevents APP from being Directly Cleaved by γ-Secretase.

BACKGROUND: Alzheimer's disease (AD) is characterized by the deposition of amyloid-ß peptide (Aß) in the brain. Aß is produced by sequential ß- and γ-secretase cleavages of amyloid precursor protein (APP). Clinical trials targeting ß- and γ-secretases have all failed, partly because of the strong side effects. The aims of this work were to determine if the direct cleavage of APP by γ-secretase inhibits Aß production, and to identify γ-cleavage-inhibiting signals within APP that can be targeted to prevent Aß generation without inhibiting any enzyme. METHODS: An APP mutant mimicking secreted APPγ was overexpressed in cells to test ß-cleavage and Aß production. APP deletion and truncation mutants were overexpressed in cells to identify the γ-secretase-inhibiting domain. The intracellular transport of the mutants was examined using immunofluorescence. Co-immunoprecipitation was performed to investigate the molecular mechanisms. RESULTS: The APP N-terminal fragment mimicking the direct γ-cleavage product was not cleaved by beta-secretase 1 to produce detectable Aß. However, in cells, the C-terminal fragments of APP longer than the last 116 residues could not be cleaved by γ-secretase in cells. No deletion mutant was cleaved by γ-secretase. C99, the direct precursor of Aß, was no longer a γ-secretase substrate when green fluorescent protein was fused to its N-terminus. The large ectodomains prevented access to γ-secretase. CONCLUSIONS: Enabling the direct γ-cleavage of APP is a new and valid strategy to reduce Aß. However, APP does not inhibit γ-cleavage via a specific inhibitory sequence in the ectodomain. Other methods to fulfill the strategy may benefit AD prevention and therapy.

Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes.

Mutations in the Contactin-associated protein-like 2 (CNTNAP2) gene are associated with autism spectrum disorder (ASD), and ectodomain shedding of the CNTNAP2 protein plays a role in its function. However, key enzymes involved in the C-terminal cleavage of CNTNAP2 remain largely unknown, and the effect of ASD-associated mutations on this process and its role in ASD pathogenesis remain elusive. In this report we showed that CNTNAP2 undergoes sequential cleavages by furin, ADAM10/17-dependent α-secretase and presenilin-dependent γ-secretase. We identified that the cleavage sites of ADAM10 and ADAM17 in CNTNAP2 locate at its C-terminal residue I79 and L96, and the main α-cleavage product C79 by ADAM10 is required for the subsequent γ-secretase cleavage to generate CNTNAP2 intracellular domain (CICD). ASD-associated CNTNAP2 mutations impair the α-cleavage to generate C79, and the inhibition leads to ASD-like repetitive and social behavior abnormalities in the Cntnap2-I1254T knock-in mice. Finally, exogenous expression of C79 improves autism-like phenotypes in the Cntnap2-I1254T knock-in and Cntnap2-/- knockout mice. This data demonstrates that the α-secretase is essential for CNTNAP2 processing and its function. Our study indicates that inhibition of the cleavage by pathogenic mutations underlies ASD pathogenesis, and upregulation of its C-terminal fragments could have therapeutical potentials for ASD treatment.

Electroacupuncture improves cognitive function in APP/PS1 mice by inhibiting oxidative stress related hippocampal neuronal ferroptosis.

BACKGROUND: Electroacupuncture, recognized as a crucial non-pharmacological therapeutic approach, has demonstrated notable efficacy in enhancing cognitive function among Alzheimer's disease (AD) patients. This study aimed to investigate the neuroprotective properties of electroacupuncture in APP/PS1 mice with AD. METHODS: A total of thirty APP/PS1 mice were randomly assigned to three groups: the Alzheimer's disease group (AD), the electroacupuncture treatment group (EA), and the ferroptosis inhibitor deferasirox treatment group (DFX). Additionally, ten C57BL/6 mice were included as a control group (Control). In the EA group, mice underwent flat needling at Baihui and Yintang, as well as point needling at Renzhong, once daily for 15 min each time. In the DFX group, mice received intraperitoneal injections of deferasirox at a dosage of 100 mg/kg/day. Following the 28-day treatment period, behavioral evaluation, morphological observation of neurons, and detection of neuronal ferroptosis were conducted. RESULTS: The electroacupuncture treatment demonstrated a significant improvement in spatial learning, memory ability, and neuronal damage in mice with AD. Analysis of neuronal ferroptosis markers indicated that electroacupuncture interventions reduced the elevated levels of malondialdehyde, iron, and ptgs2 expression, while also increasing superoxide dismutase activity, Ferroportin 1 and glutathione peroxidase 4 expression. Moreover, the regulatory impact of electroacupuncture on ferroptosis may be attributed to its ability to enhance the expression and nuclear translocation of Nrf2. CONCLUSIONS: This study suggested that electroacupuncture could inhibit the neuronal ferroptosis by activating the antioxidant function in neurons through p62/Keap1/Nrf2 signal pathway, thereby improve the cognitive function of AD mice by the neuronal protection effect.

Association of Blood MicroRNA Expression and Polymorphisms with Cognitive and Biomarker Changes in Older Adults.

BACKGROUND: Identifying individuals before the onset of overt symptoms is key in the prevention of Alzheimer's disease (AD). OBJECTIVES: Investigate the use of miRNA as early blood-biomarker of cognitive decline in older adults. DESIGN: Cross-sectional. SETTING: Two observational cohorts (CHARIOT-PRO, Alzheimer's Disease Neuroimaging Initiative (ADNI)). PARTICIPANTS: 830 individuals without overt clinical symptoms from CHARIOT-PRO and 812 individuals from ADNI. MEASUREMENTS: qPCR analysis of a prioritised set of 38 miRNAs in the blood of individuals from CHARIOT-PRO, followed by a brain-specific functional enrichment analysis for the significant miRNAs. In ADNI, genetic association analysis for polymorphisms within the significant miRNAs' genes and CSF levels of phosphorylated-tau, total-tau, amyloid-ß42, soluble-TREM2 and BACE1 activity using whole genome sequencing data. Post-hoc analysis using multi-omics datasets. RESULTS: Six miRNAs (hsa-miR-128-3p, hsa-miR-144-5p, hsa-miR-146a-5p, hsa-miR-26a-5p, hsa-miR-29c-3p and hsa-miR-363-3p) were downregulated in the blood of individuals with low cognitive performance on the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). The pathway enrichment analysis indicated involvement of apoptosis and inflammation, relevant in early AD stages. Polymorphisms within genes encoding for hsa-miR-29c-3p and hsa-miR-146a-5p were associated with CSF levels of amyloid-ß42, soluble-TREM2 and BACE1 activity, and 21 variants were eQTL for hippocampal MIR29C expression. CONCLUSIONS: six miRNAs may serve as potential blood biomarker of subclinical cognitive deficits in AD. Polymorphisms within these miRNAs suggest a possible interplay between the amyloid cascade and microglial activation at preclinical stages of AD.

Negative Regulation of Cathepsins by ß-Amyloid.

Genome wide association study (GWAS) uncovered Alzheimer's disease (AD) risk genes linked to the endo-lysosomal pathway. This pathway seems to be the gateway of protein aggregates, such as tau and α-synuclein, to the cytoplasm. Furthermore, we and others reported that the amyloid precursor protein (APP) C99 is predominantly processed by γ-secretase in the endo-lysosomal compartments, and ß-amyloid (Aß) peptides are enriched in the same subcellular loci. While the role(s) of APP/Aß in the endo-lysosomal pathway has not been fully established, a recent study reported that Aß, in particular Aß42, inhibits cathepsin D (CTSD) activity. Here, we show using a cell-free in vitro assay that Aß42 also blocks cathepsin B (CTSB) activity. Furthermore, we uncovered that the autocatalytic processing (i.e., conversion of single chain to heavy/light chains) of CTSB and CTSD is accelerated in APP-deficient cells compared with wild-type controls. Taken together, our findings further support the negative regulation of cathepsins by Aß.

Cleavage efficiency of the intramembrane protease γ-secretase is reduced by the palmitoylation of a substrate's transmembrane domain.

The intramembrane protease γ-secretase has broad physiological functions, but also contributes to Notch-dependent tumors and Alzheimer's disease. While γ-secretase cleaves numerous membrane proteins, only few nonsubstrates are known. Thus, a fundamental open question is how γ-secretase distinguishes substrates from nonsubstrates and whether sequence-based features or post-translational modifications of membrane proteins contribute to substrate recognition. Using mass spectrometry-based proteomics, we identified several type I membrane proteins with short ectodomains that were inefficiently or not cleaved by γ-secretase, including 'pituitary tumor-transforming gene 1-interacting protein' (PTTG1IP). To analyze the mechanism preventing cleavage of these putative nonsubstrates, we used the validated substrate FN14 as a backbone and replaced its transmembrane domain (TMD), where γ-cleavage occurs, with the one of nonsubstrates. Surprisingly, some nonsubstrate TMDs were efficiently cleaved in the FN14 backbone, demonstrating that a cleavable TMD is necessary, but not sufficient for cleavage by γ-secretase. Cleavage efficiencies varied by up to 200-fold. Other TMDs, including that of PTTG1IP, were still barely cleaved within the FN14 backbone. Pharmacological and mutational experiments revealed that the PTTG1IP TMD is palmitoylated, which prevented cleavage by γ-secretase. We conclude that the TMD sequence of a membrane protein and its palmitoylation can be key factors determining substrate recognition and cleavage efficiency by γ-secretase.

C-terminal amino acids in the type I transmembrane domain of L-type lectin VIP36 affect γ-secretase susceptibility.

Regulated intramembrane proteolysis (RIP) is a two-step processing mechanism for transmembrane proteins consisting of ectodomain shedding (shedding), which removes the extracellular domain through juxtamembrane processing and intramembrane proteolysis, which processes membrane-anchored shedding products within the transmembrane domain. RIP irreversibly converts one transmembrane protein into multiple soluble proteins that perform various physiological functions. The only requirement for the substrate of γ-secretase, the major enzyme responsible for intramembrane proteolysis of type I transmembrane proteins, is the absence of a large extracellular domain, and it is thought that γ-secretase can process any type I membrane protein as long as it is shed. In the present study, we showed that the shedding susceptible type I membrane protein VIP36 (36 kDa vesicular integral membrane protein) and its homolog, VIPL, have different γ-secretase susceptibilities in their transmembrane domains. Analysis of the substitution mutants suggested that γ-secretase susceptibility is regulated by C-terminal amino acids in the transmembrane domain. We also compared the transmembrane domains of several shedding susceptible membrane proteins and found that each had a different γ-secretase susceptibility. These results suggest that the transmembrane domain is not simply a stretch of hydrophobic amino acids but is an important element that regulates membrane protein function by controlling the lifetime of the membrane-anchored shedding product.

Cell-free protein production of a gamma secretase homolog.

Cleavage of the transmembrane domain (TMD) of amyloid-ß precursor protein (APP) by γ-secretase, an intramembrane aspartyl protease, generates Aß peptides of various lengths that form plaques in the brains of Alzheimer's disease patients. Although the debate has not been finally resolved whether these plaques trigger the onset of Alzheimer's or are side products, disease-related mutations suggest their implication in the etiology of the dementia. These occur both in presenilin, the catalytic subunit of γ-secretase, and in the TMD of APP. Despite two seminal cryo-electron microscopy structures that show the complex of γ-secretase with its substrates APP and Notch, the mechanism of γ-secretase is not yet fully understood. Especially on which basis it selects its substrates is still an enigma. The presenilin homolog PSH from the archaeon Methanoculleus marisnigri JR1 (MCMJR1) is catalytically active without accessory proteins in contrast to γ-secretase making it an excellent model for studies of the basic cleavage process. We here focused on the cell-free expression of PSH screening a range of conditions. Cleavage assays to verify the activity show that not only the yield, but mainly the activity of the protease depends on the careful selection of expression conditions. Optimal results were found for a cell-free expression at relatively low temperature, 20 °C, employing cell lysates prepared from E. coli Rosetta cells. To speed up protein preparation for immediate functional assays, a crude purification protocol was developed. This allows to produce ready-made PSH in a fast and efficient manner in less than two days.

Effects of DHA (omega-3 fatty acid) and estradiol on amyloid ß-peptide regulation in the brain.

In the early stages of sporadic Alzheimer's disease (SAD), there is a strong correlation between memory impairment and cortical levels of soluble amyloid-ß peptide oligomers (Aß). It has become clear that Aß disrupt glutamatergic synaptic function, which can in turn lead to the characteristic cognitive deficits of SAD, but the actual pathways are still not well understood. This opinion article describes the pathogenic mechanisms underlying cerebral amyloidosis. These mechanisms are dependent on the amyloid precursor protein and concern the synthesis of Aß peptides with competition between the non-amyloidogenic pathway and the amyloidogenic pathway (i.e. a competition between the ADAM10 and BACE1 enzymes), on the one hand, and the various processes of Aß residue clearance, on the other hand. This clearance mobilizes both endopeptidases (NEP, and IDE) and removal transporters across the blood-brain barrier (LRP1, ABCB1, and RAGE). Lipidated ApoE also plays a major role in all processes. The disturbance of these pathways induces an accumulation of Aß. The description of the mechanisms reveals two key molecules in particular: (i) free estradiol, which has genomic and non-genomic action, and (ii) free DHA as a preferential ligand of PPARα-RXRα and PPARÉ£-RXRα heterodimers. DHA and free estradiol are also self-regulating, and act in synergy. When a certain level of chronic DHA and free estradiol deficiency is reached, a permanent imbalance is established in the central nervous system. The consequences of these deficits are revealed in particular by the presence of Aß peptide deposits, as well as other markers of the etiology of SAD.

Functional and topological analysis of PSENEN, the fourth subunit of the γ-secretase complex.

The γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aß peptides in Alzheimer's disease. The complex consists of four subunits, with Presenilin harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2, encoded by the gene Psenen, in vivo and in vitro. We find a profound Notch deficiency phenotype in Psenen-/- embryos confirming the essential role of PSENEN in the γ-secretase complex. We used Psenen-/- fibroblasts to explore the structure-function of PSENEN by the scanning cysteine accessibility method. Glycine 22 and proline 27, which border the membrane domains 1 and 2 of PSENEN, are involved in complex formation and stabilization of γ-secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the γ-secretase complex and might contribute to the catalytic mechanism of the enzyme.