Structural basis of γ-secretase inhibition and modulation by small molecule drugs.

Development of γ-secretase inhibitors (GSIs) and modulators (GSMs) represents an attractive therapeutic opportunity for Alzheimer's disease (AD) and cancers. However, how these GSIs and GSMs target γ-secretase has remained largely unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human γ-secretase bound individually to two GSI clinical candidates, Semagacestat and Avagacestat, a transition state analog GSI L685,458, and a classic GSM E2012, at overall resolutions of 2.6-3.1 Å. Remarkably, each of the GSIs occupies the same general location on presenilin 1 (PS1) that accommodates the ß strand from amyloid precursor protein or Notch, interfering with substrate recruitment. L685,458 directly coordinates the two catalytic aspartate residues of PS1. E2012 binds to an allosteric site of γ-secretase on the extracellular side, potentially explaining its modulating activity. Structural analysis reveals a set of shared themes and variations for inhibitor and modulator recognition that will guide development of the next-generation substrate-selective inhibitors.

Alzheimer's-Causing Mutations Shift Aß Length by Destabilizing γ-Secretase-Aßn Interactions.

Alzheimer's disease (AD)-linked mutations in Presenilins (PSEN) and the amyloid precursor protein (APP) lead to production of longer amyloidogenic Aß peptides. The shift in Aß length is fundamental to the disease; however, the underlying mechanism remains elusive. Here, we show that substrate shortening progressively destabilizes the consecutive enzyme-substrate (E-S) complexes that characterize the sequential γ-secretase processing of APP. Remarkably, pathogenic PSEN or APP mutations further destabilize labile E-S complexes and thereby promote generation of longer Aß peptides. Similarly, destabilization of wild-type E-S complexes by temperature, compounds, or detergent promotes release of amyloidogenic Aß. In contrast, E-Aßn stabilizers increase γ-secretase processivity. Our work presents a unifying model for how PSEN or APP mutations enhance amyloidogenic Aß production, suggests that environmental factors may increase AD risk, and provides the theoretical basis for the development of γ-secretase/substrate stabilizing compounds for the prevention of AD.

Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aß Pool.

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

Cortical neurodegeneration caused by Psen1 mutations is independent of Aß.

Mutations in the PSEN genes are the major cause of familial Alzheimer's disease, and presenilin (PS) is the catalytic subunit of γ-secretase, which cleaves type I transmembrane proteins, including the amyloid precursor protein (APP) to release Aß peptides. While PS plays an essential role in the protection of neuronal survival, PSEN mutations also increase the ratio of Aß42/Aß40. Thus, it remains unresolved whether PSEN mutations cause AD via a loss of its essential function or increases of Aß42/Aß40. Here, we test whether the knockin (KI) allele of Psen1 L435F, the most severe FAD mutation located closest to the active site of γ-secretase, causes age-dependent cortical neurodegeneration independent of Aß by crossing various Psen mutant mice to the App-null background. We report that removing Aß completely through APP deficiency has no impact on the age-dependent neurodegeneration in Psen mutant mice, as shown by the absence of effects on the reduced cortical volume and decreases of cortical neurons at the ages of 12 and 18 mo. The L435F KI allele increases Aß42/Aß40 in the cerebral cortex while decreasing de novo production and steady-state levels of Aß42 and Aß40 in the presence of APP. Furthermore, APP deficiency does not alleviate elevated apoptotic cell death in the cerebral cortex of Psen mutant mice at the ages of 2, 12, and 18 mo, nor does it affect the progressive microgliosis in these mice. Our findings demonstrate that Psen1 mutations cause age-dependent neurodegeneration independent of Aß, providing further support for a loss-of-function pathogenic mechanism underlying PSEN mutations.

Directed conversion of Alzheimer's disease patient skin fibroblasts into functional neurons.

Directed conversion of mature human cells, as from fibroblasts to neurons, is of potential clinical utility for neurological disease modeling as well as cell therapeutics. Here, we describe the efficient generation of human-induced neuronal (hiN) cells from adult skin fibroblasts of unaffected individuals and Alzheimer's patients, using virally transduced transcription regulators and extrinsic support factors. hiN cells from unaffected individuals display morphological, electrophysiological, and gene expression profiles that typify glutamatergic forebrain neurons and are competent to integrate functionally into the rodent CNS. hiN cells from familial Alzheimer disease (FAD) patients with presenilin-1 or -2 mutations exhibit altered processing and localization of amyloid precursor protein (APP) and increased production of Aß, relative to the source patient fibroblasts or hiN cells from unaffected individuals. Together, our findings demonstrate directed conversion of human fibroblasts to a neuronal phenotype and reveal cell type-selective pathology in hiN cells derived from FAD patients.

The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer's disease.

Innate immunity is associated with Alzheimer's disease1, but the influence of immune activation on the production of amyloid-ß is unknown2,3. Here we identify interferon-induced transmembrane protein 3 (IFITM3) as a γ-secretase modulatory protein, and establish a mechanism by which inflammation affects the generation of amyloid-ß. Inflammatory cytokines induce the expression of IFITM3 in neurons and astrocytes, which binds to γ-secretase and upregulates its activity, thereby increasing the production of amyloid-ß. The expression of IFITM3 is increased with ageing and in mouse models that express familial Alzheimer's disease genes. Furthermore, knockout of IFITM3 reduces γ-secretase activity and the formation of amyloid plaques in a transgenic mouse model (5xFAD) of early amyloid deposition. IFITM3 protein is upregulated in tissue samples from a subset of patients with late-onset Alzheimer's disease that exhibit higher γ-secretase activity. The amount of IFITM3 in the γ-secretase complex has a strong and positive correlation with γ-secretase activity in samples from patients with late-onset Alzheimer's disease. These findings reveal a mechanism in which γ-secretase is modulated by neuroinflammation via IFITM3 and the risk of Alzheimer's disease is thereby increased.

Human Presenilin-1 delivered by AAV9 rescues impaired γ-secretase activity, memory deficits, and neurodegeneration in Psen mutant mice.

Mutations in the Presenilin (PSEN1 and PSEN2) genes are the major cause of early-onset familial Alzheimer's disease (FAD). Presenilin (PS) is the catalytic subunit of the γ-secretase complex, which cleaves type I transmembrane proteins, such as Notch and the amyloid precursor protein (APP), and plays an evolutionarily conserved role in the protection of neuronal survival during aging. FAD PSEN1 mutations exhibit impaired γ-secretase activity in cell culture, in vitro, and knockin (KI) mouse brains, and the L435F mutation is the most severe in reducing γ-secretase activity and is located closest to the active site of γ-secretase. Here, we report that introduction of the codon-optimized wild-type human PSEN1 cDNA by adeno-associated virus 9 (AAV9) results in broadly distributed, sustained, low to moderate levels of human PS1 (hPS1) expression and rescues impaired γ-secretase activity in the cerebral cortex of Psen mutant mice either lacking PS or expressing the Psen1 L435F KI allele, as evaluated by endogenous γ-secretase substrates of APP and recombinant γ-secretase products of Notch intracellular domain and Aß peptides. Furthermore, introduction of hPS1 by AAV9 alleviates impairments of synaptic plasticity and learning and memory in Psen mutant mice. Importantly, AAV9 delivery of hPS1 ameliorates neurodegeneration in the cerebral cortex of aged Psen mutant mice, as shown by the reversal of age-dependent loss of cortical neurons and elevated microgliosis and astrogliosis. These results together show that moderate hPS1 expression by AAV9 is sufficient to rescue impaired γ-secretase activity, synaptic and memory deficits, and neurodegeneration caused by Psen mutations in mouse models.

Identification of PS1/gamma-secretase and glutamate transporter GLT-1 interaction sites.

The recently discovered interaction between Presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for generating amyloid-ß peptides, and GLT-1, a major glutamate transporter in the brain (EAAT2), provides a mechanistic link between these two key factors involved in Alzheimer's disease (AD) pathology. Modulating this interaction can be crucial to understand the consequence of such crosstalk in AD context and beyond. However, the interaction sites between these two proteins are unknown. Herein, we utilized an alanine scanning approach coupled with FRET-based fluorescence lifetime imaging microscopy to identify the interaction sites between PS1 and GLT-1 in their native environment within intact cells. We found that GLT-1 residues at position 276 to 279 (TM5) and PS1 residues at position 249 to 252 (TM6) are crucial for GLT-1-PS1 interaction. These results have been cross validated using AlphaFold Multimer prediction. To further investigate whether this interaction of endogenously expressed GLT-1 and PS1 can be prevented in primary neurons, we designed PS1/GLT-1 cell-permeable peptides (CPPs) targeting the PS1 or GLT-1 binding site. We used HIV TAT domain to allow for cell penetration which was assayed in neurons. First, we assessed the toxicity and penetration of CPPs by confocal microscopy. Next, to ensure the efficiency of CPPs, we monitored the modulation of GLT-1-PS1 interaction in intact neurons by fluorescence lifetime imaging microscopy. We saw significantly less interaction between PS1 and GLT-1 with both CPPs. Our study establishes a new tool to study the functional aspect of GLT-1-PS1 interaction and its relevance in normal physiology and AD models.

Akt activation ameliorates deficits in hippocampal-dependent memory and activity-dependent synaptic protein synthesis in an Alzheimer's disease mouse model.

Protein kinase-B (Akt) and the mechanistic target of rapamycin (mTOR) signaling pathways are implicated in Alzheimer's disease (AD) pathology. Akt/mTOR signaling pathways, activated by external inputs, enable new protein synthesis at the synapse and synaptic plasticity. The molecular mechanisms impeding new protein synthesis at the synapse in AD pathogenesis remain elusive. Here, we aimed to understand the molecular mechanisms prior to the manifestation of histopathological hallmarks by characterizing Akt1/mTOR signaling cascades and new protein synthesis in the hippocampus of WT and amyloid precursor protein/presenilin-1 (APP/PS1) male mice. Intriguingly, compared to those in WT mice, we found significant decreases in pAkt1, pGSK3ß, pmTOR, pS6 ribosomal protein, and p4E-BP1 levels in both post nuclear supernatant and synaptosomes isolated from the hippocampus of one-month-old (presymptomatic) APP/PS1 mice. In synaptoneurosomes prepared from the hippocampus of presymptomatic APP/PS1 mice, activity-dependent protein synthesis at the synapse was impaired and this deficit was sustained in young adults. In hippocampal neurons from C57BL/6 mice, downregulation of Akt1 precluded synaptic activity-dependent protein synthesis at the dendrites but not in the soma. In three-month-old APP/PS1 mice, Akt activator (SC79) administration restored deficits in memory recall and activity-dependent synaptic protein synthesis. C57BL/6 mice administered with an Akt inhibitor (MK2206) resulted in memory recall deficits compared to those treated with vehicle. We conclude that dysregulation of Akt1/mTOR and its downstream signaling molecules in the hippocampus contribute to memory recall deficits and loss of activity-dependent synaptic protein synthesis. In AD mice, however, Akt activation ameliorates deficits in memory recall and activity-dependent synaptic protein synthesis.

Quantitative comparison of presenilin protein expression reveals greater activity of PS2-γ-secretase.

γ-secretase processing of amyloid precursor protein (APP) has long been of interest in the pathological progression of Alzheimer's disease (AD) due to its role in the generation of amyloid-ß. The catalytic component of the enzyme is the presenilins of which there are two homologues, Presenilin-1 (PS1) and Presenilin-2 (PS2). The field has focussed on the PS1 form of this enzyme, as it is typically considered the more active at APP processing. However, much of this work has been completed without appropriate consideration of the specific levels of protein expression of PS1 and PS2. We propose that expression is an important factor in PS1- and PS2-γ-secretase activity, and that when this is considered, PS1 does not have greater activity than PS2. We developed and validated tools for quantitative assessment of PS1 and PS2 protein expression levels to enable the direct comparison of PS in exogenous and endogenous expression systems, in HEK-293 PS1 and/or PS2 knockout cells. We show that exogenous expression of Myc-PS1-NTF is 5.5-times higher than Myc-PS2-NTF. Quantitating endogenous PS protein levels, using a novel PS1/2 fusion standard we developed, showed similar results. When the marked difference in PS1 and PS2 protein levels is considered, we show that compared to PS1-γ-secretase, PS2-γ-secretase has equal or more activity on APP and Notch1. This study has implications for understanding the PS1- and PS2-specific contributions to substrate processing, and their potential influence in AD pathogenesis.

Differential disruptions in population coding along the dorsal-ventral axis of CA1 in the APP/PS1 mouse model of Aß pathology.

Alzheimer's Disease (AD) is characterized by a range of behavioral alterations, including memory loss and psychiatric symptoms. While there is evidence that molecular pathologies, such as amyloid beta (Aß), contribute to AD, it remains unclear how this histopathology gives rise to such disparate behavioral deficits. One hypothesis is that Aß exerts differential effects on neuronal circuits across brain regions, depending on the neurophysiology and connectivity of different areas. To test this, we recorded from large neuronal populations in dorsal CA1 (dCA1) and ventral CA1 (vCA1), two hippocampal areas known to be structurally and functionally diverse, in the APP/PS1 mouse model of amyloidosis. Despite similar levels of Aß pathology, dCA1 and vCA1 showed distinct disruptions in neuronal population activity as animals navigated a virtual reality environment. In dCA1, pairwise correlations and entropy, a measure of the diversity of activity patterns, were decreased in APP/PS1 mice relative to age-matched C57BL/6 controls. However, in vCA1, APP/PS1 mice had increased pair-wise correlations and entropy as compared to age matched controls. Finally, using maximum entropy models, we connected the microscopic features of population activity (correlations) to the macroscopic features of the population code (entropy). We found that the models' performance increased in predicting dCA1 activity, but decreased in predicting vCA1 activity, in APP/PS1 mice relative to the controls. Taken together, we found that Aß exerts distinct effects across different hippocampal regions, suggesting that the various behavioral deficits of AD may reflect underlying heterogeneities in neuronal circuits and the different disruptions that Aß pathology causes in those circuits.

Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations.

Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.

Presenilin-1 ΔE9 mutation associated sarcoplasmic reticulum leak alters [Ca2+]i distribution in human iPSC-derived cardiomyocytes.

Mutations in ubiquitously expressed presenilin genes (PSENs) lead to early-onset familial Alzheimer's disease (FAD), but patients carrying the mutation also suffer from heart diseases. To elucidate the cardiac myocyte specific effects of PSEN ΔE9, we studied cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) from patients carrying AD-causing PSEN1 exon 9 deletion (PSEN1 ΔE9). When compared with their isogenic controls, PSEN1 ΔE9 cardiomyocytes showed increased sarcoplasmic reticulum (SR) Ca2+ leak that was resistant to blockage of ryanodine receptors (RyRs) by tetracaine or inositol-3-reseceptors (IP3Rs) by 2-ABP. The SR Ca2+ leak did not affect electrophysiological properties of the hiPSC-CMs, but according to experiments and in silico simulations the leak induces a diastolic buildup of [Ca2+] near the perinuclear SR and reduces the releasable Ca2+ during systole. This demonstrates that PSEN1 ΔE9 induced SR Ca2+ leak has specific effects in iPSC-CMs, reflecting their unique structural and calcium signaling features. The results shed light on the physiological and pathological mechanisms of PSEN1 in cardiac myocytes and explain the intricacies of comorbidity associated with AD-causing mutations in PSEN1.

Presenilin 1 Is a Therapeutic Target in Pulmonary Hypertension and Promotes Vascular Remodeling.

Small muscular pulmonary artery remodeling is a dominant feature of pulmonary arterial hypertension (PAH). PSEN1 affects angiogenesis, cancer, and Alzheimer's disease. We aimed to determine the role of PSEN1 in the pathogenesis of vascular remodeling in pulmonary hypertension (PH). Hemodynamics and vascular remodeling in the Psen1-knockin and smooth muscle-specific Psen1-knockout mice were assessed. The functional partners of PSEN1 were predicted by bioinformatics analysis and biochemical experiments. The therapeutic effect of PH was evaluated by administration of the PSEN1-specific inhibitor ELN318463. We discovered that both the mRNA and protein levels of PSEN1 were increased over time in hypoxic rats, monocrotaline rats, and Su5416/hypoxia mice. Psen1 transgenic mice were highly susceptible to PH, whereas smooth muscle-specific Psen1-knockout mice were resistant to hypoxic PH. STRING analysis showed that Notch1/2/3, ß-catenin, Cadherin-1, DNER (delta/notch-like epidermal growth factor-related receptor), TMP10, and ERBB4 appeared to be highly correlated with PSEN1. Immunoprecipitation confirmed that PSEN1 interacts with ß-catenin and DNER, and these interactions were suppressed by the catalytic PSEN1 mutations D257A, D385A, and C410Y. PSEN1 was found to mediate the nuclear translocation of the Notch1 intracellular domains and activated RBP-Jκ. Octaarginine-coated liposome-mediated pharmacological inhibition of PSEN1 significantly prevented and reversed the pathological process in hypoxic and monocrotaline-induced PH. PSEN1 essentially drives the pathogenesis of PAH and interacted with the noncanonical Notch ligand DNER. PSEN1 can be used as a promising molecular target for treating PAH. PSEN1 inhibitor ELN318463 can prevent and reverse the progression of PH and can be developed as a potential anti-PAH drug.

Pathogenic Aß production by heterozygous PSEN1 mutations is intrinsic to the mutant protein and not mediated by conformational hindrance of wild-type PSEN1.

Presenilin-1 (PSEN1) is the catalytic subunit of the intramembrane protease γ-secretase and undergoes endoproteolysis during its maturation. Heterozygous mutations in the PSEN1 gene cause early-onset familial Alzheimer's disease (eFAD) and increase the proportion of longer aggregation-prone amyloid-ß peptides (Aß42 and/or Aß43). Previous studies had suggested that PSEN1 mutants might act in a dominant-negative fashion by functional impediment of wild-type PSEN1, but the exact mechanism by which PSEN1 mutants promote pathogenic Aß production remains controversial. Using dual recombinase-mediated cassette exchange (dRMCE), here we generated a panel of isogenic embryonic and neural stem cell lines with heterozygous, endogenous expression of PSEN1 mutations. When catalytically inactive PSEN1 was expressed alongside the wild-type protein, we found the mutant accumulated as a full-length protein, indicating that endoproteolytic cleavage occurred strictly as an intramolecular event. Heterozygous expression of eFAD-causing PSEN1 mutants increased the Aß42/Aß40 ratio. In contrast, catalytically inactive PSEN1 mutants were still incorporated into the γ-secretase complex but failed to change the Aß42/Aß40 ratio. Finally, interaction and enzyme activity assays demonstrated the binding of mutant PSEN1 to other γ-secretase subunits, but no interaction between mutant and wild-type PSEN1 was observed. These results establish that pathogenic Aß production is an intrinsic property of PSEN1 mutants and strongly argue against a dominant-negative effect in which PSEN1 mutants would compromise the catalytic activity of wild-type PSEN1 through conformational effects.

Different transmembrane domains determine the specificity and efficiency of the cleavage activity of the γ-secretase subunit presenilin.

The γ-secretase complex catalyzes the intramembrane cleavage of C99, a carboxy-terminal fragment of the amyloid precursor protein. Two paralogs of its catalytic subunit presenilin (PS1 and PS2) are expressed which are autocatalytically cleaved into an N-terminal and a C-terminal fragment during maturation of γ-secretase. In this study, we compared the efficiency and specificity of C99 cleavage by PS1- and PS2-containing γ-secretases. Mass spectrometric analysis of cleavage products obtained in cell-free and cell-based assays revealed that the previously described lower amyloid-ß (Aß)38 generation by PS2 is accompanied by a reciprocal increase in Aß37 production. We further found PS1 and PS2 to show different preferences in the choice of the initial cleavage site of C99. However, the differences in Aß38 and Aß37 generation appear to mainly result from altered subsequent stepwise cleavage of Aß peptides. Apart from these differences in cleavage specificity, we confirmed a lower efficiency of initial C99 cleavage by PS2 using a detergent-solubilized γ-secretase system. By investigating chimeric PS1/2 molecules, we show that the membrane-embedded, nonconserved residues of the N-terminal fragment mainly account for the differential cleavage efficiency and specificity of both presenilins. At the level of individual transmembrane domains (TMDs), TMD3 was identified as a major modulator of initial cleavage site specificity. The efficiency of endoproteolysis strongly depends on nonconserved TMD6 residues at the interface to TMD2, i.e., at a putative gate of substrate entry. Taken together, our results highlight the role of individual presenilin TMDs in the cleavage of C99 and the generation of Aß peptides.

Membrane lipid remodeling modulates γ-secretase processivity.

Imbalances in the amounts of amyloid-ß peptides (Aß) generated by the membrane proteases ß- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase have shown that increasing membrane thickness modulates Aß generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aß37 and/or Aß38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aß secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies.

Selective inhibitors of the PSEN1-gamma-secretase complex.

Clinical development of γ-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer's disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific γ-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1-APH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different γ-secretase complexes might provide safer drugs in the future.

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.

The Alzheimer's disease-associated C99 fragment of APP regulates cellular cholesterol trafficking.

The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP), and how this relationship relates to Alzheimer's disease (AD) pathogenesis, is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cellular cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol levels resides. The intracellular transport of cholesterol from the PM to the ER is believed to be activated by a lipid-sensing peptide(s) in the ER that can cluster PM-derived cholesterol into transient detergent-resistant membrane domains (DRMs) within the ER, also called the ER regulatory pool of cholesterol. When formed, these cholesterol-rich domains in the ER maintain cellular homeostasis by inducing cholesterol esterification as a mechanism of detoxification while attenuating its de novo synthesis. In this manuscript, we propose that the 99-aa C-terminal fragment of APP (C99), when delivered to the ER for cleavage by γ-secretase, acts as a lipid-sensing peptide that forms regulatory DRMs in the ER, called mitochondria-associated ER membranes (MAM). Our data in cellular AD models indicates that increased levels of uncleaved C99 in the ER, an early phenotype of the disease, upregulates the formation of these transient DRMs by inducing the internalization of extracellular cholesterol and its trafficking from the PM to the ER. These results suggest a novel role for C99 as a mediator of cholesterol disturbances in AD, potentially explaining early hallmarks of the disease.