BACE1-cleavage of Sez6 and Sez6L is elevated in Niemann-Pick type C disease mouse brains.

It is intriguing that a rare, inherited lysosomal storage disorder Niemann-Pick type C (NPC) shares similarities with Alzheimer's disease (AD). We have previously reported an enhanced processing of ß-amyloid precursor protein (APP) by ß-secretase (BACE1), a key enzyme in the pathogenesis of AD, in NPC1-null cells. In this work, we characterized regional and temporal expression and processing of the recently identified BACE1 substrates seizure protein 6 (Sez6) and seizure 6-like protein (Sez6L), and APP, in NPC1-/- (NPC1) and NPC1+/+ (wt) mouse brains. We analysed 4-weeks old brains to detect the earliest changes associated with NPC, and 10-weeks of age to identify changes at terminal disease stage. Sez6 and Sez6L were selected due to their predominant cleavage by BACE1, and their potential role in synaptic function that may contribute to presentation of seizures and/or motor impairments in NPC patients. While an enhanced BACE1-cleavage of all three substrates was detected in NPC1 vs. wt-mouse brains at 4-weeks of age, at 10-weeks increased proteolysis by BACE1 was observed for Sez6L in the cortex, hippocampus and cerebellum of NPC1-mice. Interestingly, both APP and Sez6L were found to be expressed in Purkinje neurons and their immunostaining was lost upon Purkinje cell neurodegeneration in 10-weeks old NPC1 mice. Furthermore, in NPC1- vs. wt-mouse primary cortical neurons, both Sez6 and Sez6L showed increased punctuate staining within the endolysosomal pathway as well as increased Sez6L and BACE1-positive puncta. This indicates that a trafficking defect within the endolysosomal pathway may play a key role in enhanced BACE1-proteolysis in NPC disease. Overall, our findings suggest that enhanced proteolysis by BACE1 could be a part of NPC disease pathogenesis. Understanding the basic biology of BACE1 and the functional impact of cleavage of its substrates is important to better evaluate the therapeutic potential of BACE1 against AD and, possibly, NPC disease.

Synthesis and evaluation of novel amide amino-ß-lactam derivatives as cholesterol absorption inhibitors.

The ß-lactam cholesterol absorption inhibitor ezetimibe is so far the only representative of this class of compounds on the market today. The goal of this work was to synthesize new amide ezetimibe analogs from trans-3-amino-(3R,4R)-ß-lactam and to test their cytotoxicity and activity as cholesterol absorption inhibitors. We synthesized six new amide ezetimibe analogs. All new compounds exhibited low toxicity in MDCKIIwt, hNPC1L1/MDCKII and HepG2 cell lines and showed significant inhibition of cholesterol uptake in hNPC1L1/MDCKII cells. In addition, we determined the activity of the three compounds to inhibit cholesterol absorption in vivo. Our results demonstrate that these compounds considerably reduce cholesterol concentrations in liver and small intestine of mice. Thus, our newly synthesized amide ezetimibe analogs are cholesterol absorption inhibitors in vitro and in vivo.

Novel amino-ß-lactam derivatives as potent cholesterol absorption inhibitors.

Two new trans-(3R,4R)-amino-ß-lactam derivatives and their diastereoisomeric mixtures were synthesized as ezetimibe bioisosteres and tested in in vitro and in vivo experiments as novel ß-lactam cholesterol absorption inhibitors. Both compounds exhibited low cytotoxicity in MDCKII, hNPC1L1/MDCKII, and HepG2 cell lines and potent inhibitory effect in hNPC1L1/MDCKII cells. In addition, these compounds markedly reduced cholesterol absorption in mice, resulting in reduced cholesterol concentrations in plasma, liver, and intestine. We determined the crystal structure of one amino-ß-lactam derivative to establish unambiguously both the absolute and relative configuration at the new stereogenic centre C17, which was assigned to be S. The pKa values for both compounds are 9.35, implying that the amino-ß-lactam derivatives and their diastereoisomeric mixtures are in form of ammonium salt in blood and the intestine. The IC50 value for the diastereoisomeric mixture is 60 µM. In vivo, it efficiently inhibited cholesterol absorption comparable to ezetimibe.

Bidirectional links between Alzheimer's disease and Niemann-Pick type C disease.

Alzheimer's disease (AD) and Niemann-Pick type C (NPC) disease are progressive neurodegenerative diseases with very different epidemiology and etiology. AD is a common cause of dementia with a complex polyfactorial etiology, including both genetic and environmental risk factors, while NPC is a very rare autosomal recessive disease. However, the diseases share some disease-related molecular pathways, including abnormal cholesterol metabolism, and involvement of amyloid-ß (Aß) and tau pathology. Here we review recent studies on these pathological traits, focusing on studies of Aß and tau pathology in NPC, and the importance of the NPC1 gene in AD. Further studies of similarities and differences between AD and NPC may be useful to increase the understanding of both these devastating neurological diseases.

Pharmacological activation of LXRs decreases amyloid-ß levels in Niemann-Pick type C model cells.

Niemann-Pick type C disease (NPC) is an inherited disorder mainly caused by loss-of-function mutations in the NPC1 gene, that lead to intracellular cholesterol accumulation and disturbed cholesterol homeostasis. Similarly to Alzheimer's disease (AD), NPC is associated with progressive neurodegeneration and altered metabolism of amyloid precursor protein (APP). Liver X receptors (LXRs), the key transcriptional regulators of cholesterol homeostasis, were reported to play neuroprotective roles in NPC mice. We investigated the impacts of LXRs on APP metabolism in mutant CHO cells lacking the NPC1 gene (-NPC1 cells). Pharmacological activation of LXRs in -NPC1 cells tended to reduce the ratio of total secreted APP (sAPP) to full length APP (flAPP) levels and sAPPß levels as well as to increase the ratio of APP Cterminal fragments to flAPP levels, resulting in decreased levels of amyloid ß (Aß) peptides. -NPC1 cells treated with LXR agonist TO901317 (TO90) displayed a modest increase in cholesterol efflux to apolipoprotein A-I (apoA-I) but not to HDL3, or in the absence of extracellular cholesterol acceptors. The observed similar reduction of Aß levels upon TO90 treatment in the presence or in the absence of extracellular apoA-I indicated a cholesterol-efflux independent effect of TO90 on Aß levels. Furthermore, TO90 had no effect on the cholesterol synthesis rate in -NPC1 cells, while it reduced the rate of cholesterol esterification. The obtained results indicate that LXR activation may decrease Aß levels in NPC1- deficient conditions. The underlying mechanism of this action does not appear to be related to effects on cholesterol efflux or synthesis rates.

Use of cerebrospinal fluid biomarker analysis for improving Alzheimer's disease diagnosis in a non-specialized setting.

Low levels of amyloid-beta42 (Abeta42) and high total-tau (t-tau) or phosphorylated-tau (p181-tau) levels in cerebrospinal fluid (CSF) were shown to be characteristic for Alzheimer's disease (AD) patients and for mildly cognitively impaired (MCI) or non-demented individuals who will progress to AD. The goal of this study was to evaluate the benefit of CSF biomarker testing in a setting with no specialized dementia centers, in order to improve the accuracy of AD diagnosis and to identify individuals with incipient AD. Using ELISA assay we analyzed CSF Abeta42, t-tau and p181-tau levels among clinically diagnosed non-demented individuals, AD patients and individuals with uncertain dementia (n=36). CSF cut-off values of low Abeta42 (less than or equal to 530 pg/mL) and high t-tau (less than or equal to 350 pg/mL) or p181-tau (less than or equal to 52 pg/mL) were used to identify individuals with AD/MCI-CSF profile, regardless of clinical diagnosis. APOE genotyping was performed using PCR-RFLP method. In accord with previous studies we detected significantly decreased levels of CSF Abeta42 and increased tau and p181-tau levels in clinically diagnosed AD group vs. non-demented controls. CSF profiling identified individuals with a typical AD/MCI-CSF pattern in clinically referred non-demented group (9 percent) and among patients with uncertain dementia (41.7 percent). APOE epsilon4-allele was associated with the CSF biomarker changes typical for AD. This study shows that in a non-specialized setting CSF biomarker testing may be used as a screening tool for improving the accuracy of AD diagnosis and for predicting individuals with incipient Alzheimer's disease who need to receive further clinical follow-up.

Amyloid-ß metabolism in Niemann-Pick C disease models and patients.

Niemann-Pick type C (NPC) is a progressive neurodegenerative lysosomal disease with altered cellular lipid trafficking. The metabolism of amyloid-ß (Aß) - previously mainly studied in Alzheimer's disease - has been suggested to be altered in NPC. Here we aimed to perform a detailed characterization of metabolic products from the amyloid precursor protein (APP) in NPC models and patients. We used multiple analytical technologies, including immunoassays and immunoprecipitation followed by mass spectrometry (IP-MS) to characterize Aß peptides and soluble APP fragments (sAPP-α/ß) in cell media from pharmacologically (U18666A) and genetically (NPC1 ( -/- ) ) induced NPC cell models, and cerebrospinal fluid (CSF) from NPC cats and human patients. The pattern of Aß peptides and sAPP-α/ß fragments in cell media was differently affected by NPC-phenotype induced by U18666A treatment and by NPC1 ( -/- ) genotype. U18666A treatment increased the secreted media levels of sAPP-α, AßX-40 and AßX-42 and reduced the levels of sAPP-ß, Aß1-40 and Aß1-42, while IP-MS showed increased relative levels of Aß5-38 and Aß5-40 in response to treatment. NPC1 ( -/- ) cells had reduced media levels of sAPP-α and Aß1-16, and increased levels of sAPP-ß. NPC cats had altered CSF distribution of Aß peptides compared with normal cats. Cats treated with the potential disease-modifying compound 2-hydroxypropyl-ß-cyclodextrin had increased relative levels of short Aß peptides including Aß1-16 compared with untreated cats. NPC patients receiving ß-cyclodextrin had reduced levels over time of CSF Aß1-42, AßX-38, AßX-40, AßX-42 and sAPP-ß, as well as reduced levels of the axonal damage markers tau and phosphorylated tau. We conclude that NPC models have altered Aß metabolism, but with differences across experimental systems, suggesting that NPC1-loss of function, such as in NPC1 ( -/- ) cells, or NPC1-dysfunction, seen in NPC patients and cats as well as in U18666A-treated cells, may cause subtle but different effects on APP degradation pathways. The preliminary findings from NPC cats suggest that treatment with cyclodextrin may have an impact on APP processing pathways. CSF Aß, sAPP and tau biomarkers were dynamically altered over time in human NPC patients.

Cholesterol-depletion corrects APP and BACE1 misstrafficking in NPC1-deficient cells.

Cholesterol accumulation in Niemann-Pick type C disease (NPC) causes increased levels of the amyloid-precursor-protein C-terminal fragments (APP-CTFs) and intracellular amyloid-ß peptide (Aß), the two central molecules in Alzheimer's disease (AD) pathogenesis. We previously reported that cholesterol accumulation in NPC-cells leads to cholesterol-dependent increased APP processing by ß-secretase (BACE1) and decreased APP expression at the cell surface (Malnar et al. Biochim Biophys Acta. 1802 (2010) 682-691.). We hypothesized that increased formation of APP-CTFs and Aß in NPC disease is due to cholesterol-mediated altered endocytic trafficking of APP and/or BACE1. Here, we show that APP endocytosis is prerequisite for enhanced Aß levels in NPC-cells. Moreover, we observed that NPC cells show cholesterol dependent sequestration and colocalization of APP and BACE1 within enlarged early/recycling endosomes which can lead to increased ß-secretase processing of APP. We demonstrated that increased endocytic localization of APP in NPC-cells is likely due to both its increased internalization and its decreased recycling to the cell surface. Our findings suggest that increased cholesterol levels, such as in NPC disease and sporadic AD, may be the upstream effector that drives amyloidogenic APP processing characteristic for Alzheimer's disease by altering endocytic trafficking of APP and BACE1.

Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.

Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between ß-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/-. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

Biochemical, neuropathological, and neuroimaging characteristics of early-onset Alzheimer's disease due to a novel PSEN1 mutation.

Familial Alzheimer's disease (AD) due to PSEN1 mutations provides an opportunity to examine AD biomarkers in persons in whom the diagnosis is certain. We describe a 55 year-old woman with clinically probable AD and a novel PSEN1 mutation who underwent genetic, clinical, biochemical and magnetic resonance and nuclear imaging assessments. We also describe neuropathological findings in her similarly affected brother. Neuropsychological testing confirmed deficits in memory, visuospatial and language function. CSF t-tau and p-tau181 were markedly elevated and Aß(42) levels reduced. FDG-PET revealed hypometabolism in the left parietotemporal cortex. FDDNP-PET showed increased binding of tracer in medial temporal and parietal lobes and in the head of the caudate and anterior putamen bilaterally. Neuropathological examination of her brother showed the typical findings of AD and the striatum demonstrated amyloid pathology and marked neurofibrillary pathology beyond that typically seen in late-onset AD. A novel S212Y substitution in PSEN1 was present in the index patient and her affected brother but not in an older unaffected sister. An in vitro assay in which the S212Y mutation was introduced in cell culture confirmed that it was associated with increased production of Aß(42). We describe biochemical, imaging, and neuropathological changes in a pedigree with a novel PSEN1 mutation. This allows us to validate the pathogenicity of this mutation and the indices used to assess AD.

Niemann-Pick type C cells show cholesterol dependent decrease of APP expression at the cell surface and its increased processing through the beta-secretase pathway.

The link between cholesterol and Alzheimer's disease has recently been revealed in Niemann-Pick type C disease. We found that NPC1(-/-) cells show decreased expression of APP at the cell surface and increased processing of APP through the beta-secretase pathway resulting in increased C99, sAPPbeta and intracellular Abeta40 levels. This effect is dependent on increased cholesterol levels, since cholesterol depletion reversed cell surface APP expression and lowered Abeta/C99 levels in NPC1(-)(/)(-) cells to the levels observed in wt cells. Finding that overexpression of C99, a direct gamma-secretase substrate, does not lead to increased intracellular Abeta levels in NPC1(-)(/)(-) cells vs. CHOwt suggests that the effect on intracellular Abeta upon cholesterol accumulation in NPC1(-)(/)(-) cells is not due to increased APP cleavage by gamma-secretase. Our results indicate that cholesterol may modulate APP processing indirectly by modulating APP expression at the cell surface and thus its cleavage by beta-secretase.

Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts.

It has been suggested that cholesterol may modulate amyloid-beta (Abeta) formation, a causative factor of Alzheimer's disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD (beta-amyloid precursor protein (APP), beta-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/Abeta formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1(-/-) cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, gamma-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP-CTF and its increased processing towards Abeta occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer's disease and supports the role of lipid rafts in these processes.