Silva Hećimović

A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis.

Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent γ-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimers disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog γ-secretase inhibitor. We propose that γ-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.

Oxidative stress parameters in plasma of Huntington's disease patients, asymptomatic Huntington’s disease gene carriers and healthy subjects

BackgroundAnimal data and postmortem studies suggest a role of oxidative stress in the Huntingtons disease (HD), but in vivo human studies have been scarce.AimTo assess the presence of oxidative stress in HD patients and its occurrence relative to clinical symptoms.MethodsOxidative stress markers were determined in plasma of HD patients (n = 19), asymptomatic HD gene carriers (with > 38 CAG repeats) (n = 11) and their respective sex and agematched healthy controls (n = 47 and n = 22) in a cross-sectional study.ResultsWith adjustment for age and sex, HD patients had higher plasma lipid peroxidation (LP) levels (ratio 1.20, 95% CI 1.09 to 1.32, p < 0.001) and lower reduced glutathione (GSH) levels (ratio 0.72, CI 0.55 to 0.94, p = 0.011) than their age and sex-matched controls. Although considerably younger, HD gene carriers did not differ from HD patients regarding LP and GSH levels, and had higher plasma LP (ratio 1.16, CI 1.02 to 1.32, p = 0.016) and lower GSH than their matched controls (ratio 0.73, CI 0.5 to 1.05). They had higher LP (ratio 1.18, CI 1.02 to 1.34, p = 0.019) and lower GSH (ratio 0.75, CI 0.51 to 1.11) than the healthy subjects matched to HD patients.ConclusionsOxidative stress is more pronounced in HD patients and asymptomatic HD gene carriers than in healthy subjects. Differences in plasma LP and GSH are in line with the brain findings in animal models of HD. Data suggest that oxidative stress occurs before the onset of the HD symptoms.

Phospholipids and Alzheimer's Disease: Alterations, Mechanisms and Potential Biomarkers

Brain is one of the richest organs in lipid content. Phospholipids (glycerophospholipids and sphingolipids) are important building blocks of cell membranes, which provide an optimal environment for protein interactions, trafficking and function. Because of that, alterations in their cellular levels could lead to different pathogenic processes in the brain, such as in Alzheimer’s disease (AD), the most common type of dementia among older populations. There is increasing evidence that phospholipid changes occur during pathogenic processes in AD. It is known that lipids are tightly connected with metabolism of the Amyloid Precursor Protein (APP), which produces Amyloid-beta peptide (Aβ), the main component of senile plaques, which represent the main pathological hallmark of AD. However, the mechanism(s) of the lipid-effect on Aβ metabolism and AD pathogenesis is still not completely understood. This review summarizes the current knowledge on phospholipid changes occurring during normal aging and discusses phospholipid changes in the human brain associated with different stages of AD, as well changes in the cerebrospinal fluid and blood/plasma, which are interesting potential biomarkers for AD diagnosis and disease monitoring. At the end, we have discussed future perspectives of phospholipid changes as potential biomarkers and as targets for development of novel treatment strategies against AD.

Mutations in APP have independent effects on Aβ and CTFγ generation

Understanding the molecular mechanism of beta-amyloid (Abeta) generation is crucial for Alzheimers disease pathogenesis as well as for normal APP function. The transmembrane domain (TM) of APP appears to undergo presenilin-dependent gamma-secretase cleavage at two topologically distinct sites: a site in the middle of the TM domain that is crucial for the generation of Abeta-peptides, and a site close to the cytoplasmic border (S3-like/epsilon site) of the TM domain that leads to production of the APP intracellular domain (CTFgamma/AICD). We demonstrate that, in contrast to the unique effect of familial Alzheimers disease (FAD) mutations in APP on Abeta42 production, some but not all FAD mutations also affect CTFgamma generation. Furthermore, changes in total CTFgamma levels do not correlate with either an increase or a decrease of any Abeta species, and inhibition of Abeta-peptide formation starting from position +1 (Abeta1-x) does not affect CTFgamma production. These results suggest that cleavage at the gamma40/42- and the S3-like sites can be dissociated, and that APP signaling and Abeta production are not tightly linked.

Expand Long PCR for fragile X mutation detection.

Fragile X mutation detection by DNA analysis enables accurate diagnosis of the fragile X syndrome. The mutation involves the expansion of CGG repeats in the FMR1 gene and has been primarily detected by the Southern blotting method. In this study we present a novel, efficient and reliable PCR protocol that is more convenient for routine diagnosis of the fragile X syndrome. This method is based on the use of the Expand Long PCR System, which enables the amplification of normal, premutated and full‐mutated alleles, and therefore provides complete CGG repeat analysis of the FMR1 gene. Normal alleles were easily detected by ethidium bromide staining of the agarose gels, suggesting that this assay could be used as a screening test for a large number of referrals. The amplified premutations and full mutations were identified by hybridization with a digoxigenin‐labeled 5′‐(CGG)5–3′ probe, followed by chemiluminescent detection. The accuracy of our Expand Long PCR protocol was confirmed by Southern blot analysis, illustrating that the Expand Long PCR results concur with those of Southern blotting. In this paper we propose a new strategy for molecular diagnosis of the fragile X syndrome in which our Expand Long PCR assay is used as the first screening test for fragile X mutation detection.

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 Alzheimers 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 Alzheimers disease and supports the role of lipid rafts in these processes.

Nano-HPLC–MS analysis of phospholipids in cerebrospinal fluid of Alzheimer’s disease patients—a pilot study

There is emerging evidence that lipids play an important role in many neurodegenerative processes, for example in Alzheimer’s disease (AD). Although different lipid alterations in the AD brain have been reported, there have only been very few investigations of lipid changes in the cerebrospinal fluid (CSF). Recent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrixes. In this study we developed an on-line HPLC–MS method for phospholipid profiling in the CSF based on nano-HPLC separation using an Amide column and detection with electrospray (ESI) quadrupole–time of flight (QTOF) MS. We achieved good separation, reproducibility, and sensitivity in monitoring of the major phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), and sphingomyelin (SM) in CSF. To emphasize the applicability of the method, a pilot study was performed on a group of CSF samples (N = 16) from individuals with probable AD and non-demented controls. We observed a statistically significant increase of SM levels (24.3 ± 2.4%) in CSF from probable AD individuals vs. controls. Our findings indicate that SM levels in the CSF could potentially provide a new lead in AD biomarker research, and show the potential of the method for disease-associated CSF phospholipid screening.

Elevated cerebrospinal fluid sphingomyelin levels in prodromal Alzheimer's disease

Alzheimers disease (AD) is the most common neurodegenerative disorder, but still without known disease mechanism, proper treatment and efficient diagnostic tools for an early stage diagnosis. There is increasing evidence that lipids, especially cholesterol and sphingolipids, may play a role in pathological processes that occur in the AD brain even in very early stages of the disease. However, lipid changes in cerebrospinal fluid (CSF) of individuals with AD have not been well studied. In previous work, we developed a reproducible and sensitive nano-HPLC-MS method for CSF phospholipids screening and conducted a pilot study to find potential phospholipid changes in CSF from individuals with AD dementia. We observed a slight increase (24%) of sphingomyelin (SM) in CSF samples from patients with probable AD compared to non-demented controls. The goal of this work was to validate our findings and to analyze how SM CSF levels change in different stages of AD from prodromal to mild and moderate AD. We found significantly increased SM levels (50.4±11.2%, p=0.003) in the CSF from individuals with prodromal AD compared to cognitively normal controls, but no change in CSF SM levels between mild and moderate AD groups and cognitively normal controls. These results suggest that alterations in the SM metabolism may contribute to early pathological processes leading to AD.

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

Alzheimers 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-β (Aβ) and tau pathology. Here we review recent studies on these pathological traits, focusing on studies of Aβ 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.

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

Familial Alzheimers 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 Aβ(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 Aβ(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.