Publications by authors named "Lucia Chávez-Gutiérrez"

35 Publications

A novel presenilin 1 duplication mutation (Ile168dup) causing Alzheimer's disease associated with myoclonus, seizures and pyramidal features.

Neurobiol Aging 2021 Feb 5. Epub 2021 Feb 5.

Dementia Research Centre, UCL Queen Square Institute of Neurology, London, UK; UK Dementia Research Institute at UCL, London, UK. Electronic address:

Mutations in the Presenilin 1 (PSEN1) gene are the most common cause of autosomal dominant familial Alzheimer's disease. We report the clinical, imaging and postmortem findings of kindred carrying a novel duplication mutation (Ile168dup) in the PSEN1 gene. We interpret the pathogenicity of this novel variant and discuss the additional neurological features (pyramidal dysfunction, myoclonus and seizures) that accompanied cognitive decline. This report broadens the clinical phenotype of PSEN1 insertion mutations while also highlighting the importance of considering duplication, insertion and deletion mutations in cases of young onset dementia.
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http://dx.doi.org/10.1016/j.neurobiolaging.2021.01.032DOI Listing
February 2021

TrkA-mediated endocytosis of p75-CTF prevents cholinergic neuron death upon γ-secretase inhibition.

Life Sci Alliance 2021 04 3;4(4). Epub 2021 Feb 3.

Molecular Basis of Neurodegeneration Unit, Institute of Biomedicine of València (IBV-CSIC), València, Spain

γ-secretase inhibitors (GSI) were developed to reduce the generation of Aβ peptide to find new Alzheimer's disease treatments. Clinical trials on Alzheimer's disease patients, however, showed several side effects that worsened the cognitive symptoms of the treated patients. The observed side effects were partially attributed to Notch signaling. However, the effect on other γ-secretase substrates, such as the p75 neurotrophin receptor (p75NTR) has not been studied in detail. p75NTR is highly expressed in the basal forebrain cholinergic neurons (BFCNs) during all life. Here, we show that GSI treatment induces the oligomerization of p75CTF leading to the cell death of BFCNs, and that this event is dependent on TrkA activity. The oligomerization of p75CTF requires an intact cholesterol recognition sequence (CRAC) and the constitutive binding of TRAF6, which activates the JNK and p38 pathways. Remarkably, TrkA rescues from cell death by a mechanism involving the endocytosis of p75CTF. These results suggest that the inhibition of γ-secretase activity in aged patients, where the expression of TrkA in the BFCNs is already reduced, could accelerate cholinergic dysfunction and promote neurodegeneration.
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http://dx.doi.org/10.26508/lsa.202000844DOI Listing
April 2021

Prodromal Alzheimer's Disease: Constitutive Upregulation of Neuroglobin Prevents the Initiation of Alzheimer's Pathology.

Front Neurosci 2020 3;14:562581. Epub 2020 Dec 3.

Molecular Neuropathology, Physiological and Pathological Processes, Centro de Biología Molecular Severo Ochoa, CSIC/UAM, Madrid, Spain.

In humans, a considerable number of the autopsy samples of cognitively normal individuals aged between 57 and 102 years have revealed the presence of amyloid plaques, one of the typical signs of AD, indicating that many of us use mechanisms that defend ourselves from the toxic consequences of Aß. The human APP NL/F (hAPP NL/F) knockin mouse appears as the ideal mouse model to identify these mechanisms, since they have high Aß42 levels at an early age and moderate signs of disease when old. Here we show that in these mice, the brain levels of the hemoprotein Neuroglobin (Ngb) increase with age, in parallel with the increase in Aß42. , in wild type neurons, exogenous Aß increases the expression of Ngb and Ngb over-expression prevents Aß toxicity. , in old hAPP NL/F mice, Ngb knockdown leads to dendritic tree simplification, an early sign of Alzheimer's disease. These results could indicate that Alzheimer's symptoms may start developing at the time when defense mechanisms start wearing out. In agreement, analysis of plasma Ngb levels in aged individuals revealed decreased levels in those whose cognitive abilities worsened during a 5-year longitudinal follow-up period.
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http://dx.doi.org/10.3389/fnins.2020.562581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744294PMC
December 2020

A multifactorial model of pathology for age of onset heterogeneity in familial Alzheimer's disease.

Acta Neuropathol 2021 02 14;141(2):217-233. Epub 2020 Dec 14.

Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Presenilin-1 (PSEN1) mutations cause familial Alzheimer's disease (FAD) characterized by early age of onset (AoO). Examination of a large kindred harboring the PSEN1-E280A mutation reveals a range of AoO spanning 30 years. The pathophysiological drivers and clinical impact of AoO variation in this population are unknown. We examined brains of 23 patients focusing on generation and deposition of beta-amyloid (Aβ) and Tau pathology profile. In 14 patients distributed at the extremes of AoO, we performed whole-exome capture to identify genotype-phenotype correlations. We also studied kinome activity, proteasome activity, and protein polyubiquitination in brain tissue, associating it with Tau phosphorylation profiles. PSEN1-E280A patients showed a bimodal distribution for AoO. Besides AoO, there were no clinical differences between analyzed groups. Despite the effect of mutant PSEN1 on production of Aβ, there were no relevant differences between groups in generation and deposition of Aβ. However, differences were found in hyperphosphorylated Tau (pTau) pathology, where early onset patients showed severe pathology with diffuse aggregation pattern associated with increased activation of stress kinases. In contrast, late-onset patients showed lesser pTau pathology and a distinctive kinase activity. Furthermore, we identified new protective genetic variants affecting ubiquitin-proteasome function in early onset patients, resulting in higher ubiquitin-dependent degradation of differentially phosphorylated Tau. In PSEN1-E280A carriers, altered γ-secretase activity and resulting Aβ accumulation are prerequisites for early AoO. However, Tau hyperphosphorylation pattern, and its degradation by the proteasome, drastically influences disease onset in individuals with otherwise similar Aβ pathology, hinting toward a multifactorial model of disease for FAD. In sporadic AD (SAD), a wide range of heterogeneity, also influenced by Tau pathology, has been identified. Thus, Tau-induced heterogeneity is a common feature in both AD variants, suggesting that a multi-target therapeutic approach should be used to treat AD.
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http://dx.doi.org/10.1007/s00401-020-02249-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847436PMC
February 2021

Computational Analysis of Alzheimer Amyloid Plaque Composition in 2D- and Elastically Reconstructed 3D-MALDI MS Images.

Anal Chem 2020 11 14;92(21):14484-14493. Epub 2020 Oct 14.

Center for Mass Spectrometry and Optical Spectroscopy (CeMOS), Mannheim University of Applied Sciences, Paul-Wittsack Str. 10, Mannheim 68163, Germany.

MALDI mass spectrometry imaging (MSI) enables label-free, spatially resolved analysis of a wide range of analytes in tissue sections. Quantitative analysis of MSI datasets is typically performed on single pixels or manually assigned regions of interest (ROIs). However, many sparse, small objects such as Alzheimer's disease (AD) brain deposits of amyloid peptides called plaques are neither single pixels nor ROIs. Here, we propose a new approach to facilitate the comparative computational evaluation of amyloid plaque-like objects by MSI: a fast PLAQUE PICKER tool that enables a statistical evaluation of heterogeneous amyloid peptide composition. Comparing two AD mouse models, APP NL-G-F and APP PS1, we identified distinct heterogeneous plaque populations in the NL-G-F model but only one class of plaques in the PS1 model. We propose quantitative metrics for the comparison of technical and biological MSI replicates. Furthermore, we reconstructed a high-accuracy 3D-model of amyloid plaques in a fully automated fashion, employing rigid and elastic MSI image registration using structured and plaque-unrelated reference ion images. Statistical single-plaque analysis in reconstructed 3D-MSI objects revealed the Aβ peptide to be located either in the core of larger plaques or in small plaques without colocalization of other Aβ isoforms. In 3D, a substantially larger number of small plaques were observed than that indicated by the 2D-MSI data, suggesting that quantitative analysis of molecularly diverse sparsely-distributed features may benefit from 3D-reconstruction. Data are available via ProteomeXchange with identifier PXD020824.
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http://dx.doi.org/10.1021/acs.analchem.0c02585DOI Listing
November 2020

Super-resolution microscopy reveals majorly mono- and dimeric presenilin1/γ-secretase at the cell surface.

Elife 2020 07 7;9. Epub 2020 Jul 7.

Laboratory for Membrane Trafficking, VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium.

γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.
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http://dx.doi.org/10.7554/eLife.56679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340497PMC
July 2020

Mechanisms of neurodegeneration - Insights from familial Alzheimer's disease.

Semin Cell Dev Biol 2020 09 14;105:75-85. Epub 2020 May 14.

VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium; Department of Neurosciences, Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, Leuven, Belgium.

The rising prevalence of Alzheimer's disease (AD), together with the lack of effective treatments, portray it as one of the major health challenges of our times. Untangling AD implies advancing the knowledge of the biology that gets disrupted during the disease while deciphering the molecular and cellular mechanisms leading to AD-related neurodegeneration. In fact, a solid mechanistic understanding of the disease processes stands as an essential prerequisite for the development of safe and effective treatments. Genetics has provided invaluable clues to the genesis of the disease by revealing deterministic genes - Presenilins (PSENs) and the Amyloid Precursor Protein (APP) - that, when affected, lead in an autosomal dominant manner to early-onset, familial AD (FAD). PSEN is the catalytic subunit of the membrane-embedded γ-secretase complexes, which act as proteolytic switches regulating key cell signalling cascades. Importantly, these intramembrane proteases are responsible for the production of Amyloid β (Aβ) peptides from APP. The convergence of pathogenic mutations on one functional pathway, the amyloidogenic cleavage of APP, strongly supports the significance of this process in AD pathogenesis. Here, we review and discuss the state-of-the-art knowledge of the molecular mechanisms underlying FAD, their implications for the sporadic form of the disease and for the development of safe AD therapeutics.
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http://dx.doi.org/10.1016/j.semcdb.2020.03.005DOI Listing
September 2020

Clinical Association of White Matter Hyperintensities Localization in a Mexican Family with Spastic Paraparesis Carrying the PSEN1 A431E Mutation.

J Alzheimers Dis 2020 ;73(3):1075-1083

Department of Molecular Biomedicine, Center for Research and Advanced Studies (CINVESTAV), CDMX, México.

Presenilin 1 gene (PSEN1) mutations are the most common cause of familial Alzheimer's disease (FAD). One of the most abundant FAD mutations, PSEN1 A431E, has been reported to be associated with spastic paraparesis in about half of its carriers, but the determining mechanisms of this phenotype are still unknown. In our study we characterized three A431E mutation carriers, one symptomatic and two asymptomatic, from a Mexican family with a history of spastic paraparesis in all of its affected members. At cognitive assessment and MRI, the symptomatic subject showed an atypical non-amnestic mild cognitive impairment with visuospatial deficits, olfactory dysfunction and significant parieto-occipital brain atrophy. Furthermore, we found several periventricular white matter hyperintensities whose progression pattern and localization correlated with their motor impairment, cognitive profile, and non-motor symptoms. Together, our data suggests that in this family the A431E mutation leads to a divergent neurological disorder in which cognitive deterioration was clinically exceeded by motor impairment and that it involves early glial and vascular pathological changes.
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http://dx.doi.org/10.3233/JAD-190978DOI Listing
January 2020

Extracellular interface between APP and Nicastrin regulates Aβ length and response to γ-secretase modulators.

EMBO J 2019 06 20;38(12). Epub 2019 May 20.

VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium

γ-Secretase complexes (GSECs) are multimeric membrane proteases involved in a variety of physiological processes and linked to Alzheimer's disease (AD). Presenilin (PSEN, catalytic subunit), Nicastrin (NCT), Presenilin Enhancer 2 (PEN-2), and Anterior Pharynx Defective 1 (APH1) are the essential subunits of GSECs. Mutations in PSEN and the Amyloid Precursor Protein (APP) cause early-onset AD GSECs successively cut APP to generate amyloid-β (Aβ) peptides of various lengths. AD-causing mutations destabilize GSEC-APP/Aβ interactions and thus enhance the production of longer Aβs, which elicit neurotoxic events underlying pathogenesis. Here, we investigated the molecular strategies that anchor GSEC and APP/Aβ during the sequential proteolysis. Our studies reveal that a direct interaction between NCT ectodomain and APP influences the stability of GSEC-Aβn assemblies and thereby modulates Aβ length. The data suggest a potential link between single-nucleotide variants in and AD risk. Furthermore, our work indicates that an extracellular interface between the protease (NCT, PSEN) and the substrate (APP) represents the target for compounds (GSMs) modulating Aβ length. Our findings may guide future rationale-based drug discovery efforts.
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http://dx.doi.org/10.15252/embj.2019101494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6576158PMC
June 2019

Trisomy of human chromosome 21 enhances amyloid-β deposition independently of an extra copy of APP.

Brain 2018 08;141(8):2457-2474

Department of Neurodegenerative Disease, UCL Institute of Neurology, London, WC1N 3BG UK.

Down syndrome, caused by trisomy of chromosome 21, is the single most common risk factor for early-onset Alzheimer's disease. Worldwide approximately 6 million people have Down syndrome, and all these individuals will develop the hallmark amyloid plaques and neurofibrillary tangles of Alzheimer's disease by the age of 40 and the vast majority will go on to develop dementia. Triplication of APP, a gene on chromosome 21, is sufficient to cause early-onset Alzheimer's disease in the absence of Down syndrome. However, whether triplication of other chromosome 21 genes influences disease pathogenesis in the context of Down syndrome is unclear. Here we show, in a mouse model, that triplication of chromosome 21 genes other than APP increases amyloid-β aggregation, deposition of amyloid-β plaques and worsens associated cognitive deficits. This indicates that triplication of chromosome 21 genes other than APP is likely to have an important role to play in Alzheimer's disease pathogenesis in individuals who have Down syndrome. We go on to show that the effect of trisomy of chromosome 21 on amyloid-β aggregation correlates with an unexpected shift in soluble amyloid-β 40/42 ratio. This alteration in amyloid-β isoform ratio occurs independently of a change in the carboxypeptidase activity of the γ-secretase complex, which cleaves the peptide from APP, or the rate of extracellular clearance of amyloid-β. These new mechanistic insights into the role of triplication of genes on chromosome 21, other than APP, in the development of Alzheimer's disease in individuals who have Down syndrome may have implications for the treatment of this common cause of neurodegeneration.
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http://dx.doi.org/10.1093/brain/awy159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061702PMC
August 2018

Modulation of γ- and β-Secretases as Early Prevention Against Alzheimer's Disease.

Biol Psychiatry 2018 02 10;83(4):320-327. Epub 2017 Aug 10.

KU Leuven Department for Neurosciences, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium.

The genetic evidence implicating amyloid-β in the initial stage of Alzheimer's disease is unequivocal. However, the long biochemical and cellular prodromal phases of the disease suggest that dementia is the result of a series of molecular and cellular cascades whose nature and connections remain unknown. Therefore, it is unlikely that treatments directed at amyloid-β will have major clinical effects in the later stages of the disease. We discuss the two major candidate therapeutic targets to lower amyloid-β in a preventive mode, i.e., γ- and β-secretase; the rationale behind these two targets; and the current state of the field.
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http://dx.doi.org/10.1016/j.biopsych.2017.08.001DOI Listing
February 2018

Alzheimer's-Causing Mutations Shift Aβ Length by Destabilizing γ-Secretase-Aβn Interactions.

Cell 2017 Jul;170(3):443-456.e14

KU Leuven-VIB Center for Brain & Disease Research, VIB, 3000 Leuven, Belgium; Department of Neurosciences, Leuven Institute for Neuroscience and Disease, KU Leuven, 3000 Leuven, Belgium. Electronic address:

Alzheimer's disease (AD)-linked mutations in Presenilins (PSEN) and the amyloid precursor protein (APP) lead to production of longer amyloidogenic Aβ peptides. The shift in Aβ 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 Aβ peptides. Similarly, destabilization of wild-type E-S complexes by temperature, compounds, or detergent promotes release of amyloidogenic Aβ. In contrast, E-Aβ stabilizers increase γ-secretase processivity. Our work presents a unifying model for how PSEN or APP mutations enhance amyloidogenic Aβ 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.
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http://dx.doi.org/10.1016/j.cell.2017.07.004DOI Listing
July 2017

Inactivation of γ-secretases leads to accumulation of substrates and non-Alzheimer neurodegeneration.

EMBO Mol Med 2017 08;9(8):1088-1099

VIB Center for Brain and Disease Research, Leuven, Belgium

γ-Secretases are a family of intramembrane cleaving aspartyl proteases and important drug targets in Alzheimer's disease. Here, we generated mice deficient for all γ-secretases in the pyramidal neurons of the postnatal forebrain by deleting the three anterior pharynx defective 1 () subunits (). The mice show progressive cortical atrophy, neuronal loss, and gliosis. Interestingly, this is associated with more than 10-fold accumulation of membrane-bound fragments of App, Aplp1, Nrg1, and Dcc, while other known substrates of γ-secretase such as Aplp2, Lrp1, and Sdc3 accumulate to lesser extents. Despite numerous reports linking neurodegeneration to accumulation of membrane-bound App fragments, deletion of App expression in the combined knockout does not rescue this phenotype. Importantly, knockout of only Aph1a- or Aph1bc-secretases causes limited and differential accumulation of substrates. This was not associated with neurodegeneration. Further development of selective Aph1-γ-secretase inhibitors should be considered for treatment of Alzheimer's disease.
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http://dx.doi.org/10.15252/emmm.201707561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538297PMC
August 2017

Identification and characterization of Nanobodies targeting the EphA4 receptor.

J Biol Chem 2017 07 19;292(27):11452-11465. Epub 2017 May 19.

From the KU Leuven-University of Leuven, Department of Neurosciences, Experimental Neurology and Leuven Research Institute for Neuroscience and Disease (LIND), 3000 Leuven, Belgium,

The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. We identified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb 39 and Nb 53 inhibited ephrin-induced phosphorylation of the EphA4 protein in a cell-based assay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3. Our results demonstrate the potential of Nanobodies to target the ligand-binding domain of EphA4. These Nanobodies may deserve further evaluation as potential therapeutics in disorders in which EphA4-mediated signaling plays a role.
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http://dx.doi.org/10.1074/jbc.M116.774141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500810PMC
July 2017

Deletion of exons 9 and 10 of the Presenilin 1 gene in a patient with Early-onset Alzheimer Disease generates longer amyloid seeds.

Neurobiol Dis 2017 Aug 28;104:97-103. Epub 2017 Apr 28.

Normandie Univ, UNIROUEN, Inserm U1245, Rouen University Hospital, Department of Genetics and CNR-MAJ, Normandy Center for Genomic and Personalized Medicine, F 76000 Rouen, France. Electronic address:

Presenilin 1 (PSEN1) mutations are the main cause of autosomal dominant Early-onset Alzheimer Disease (EOAD). Among them, deletions of exon 9 have been reported to be associated with a phenotype of spastic paraparesis. Using exome data from a large sample of 522 EOAD cases and 584 controls to search for genomic copy-number variations (CNVs), we report here a novel partial, in-frame deletion of PSEN1, removing both exons 9 and 10. The patient presented with memory impairment associated with spastic paraparesis, both starting from the age of 56years. He presented a positive family history of EOAD. We performed functional analysis to elucidate the impact of this novel deletion on PSEN1 activity as part of the γ-secretase complex. The deletion does not affect the assembly of a mature protease complex but has an extreme impact on its global endopeptidase activity. The mutant carboxypeptidase-like activity is also strongly impaired and the deleterious mutant effect leads to an incomplete digestion of long Aβ peptides and enhances the production of Aβ43, which has been shown to be potently amyloidogenic and neurotoxic in vivo.
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http://dx.doi.org/10.1016/j.nbd.2017.04.020DOI Listing
August 2017

Clinical phenotype and genetic associations in autosomal dominant familial Alzheimer's disease: a case series.

Lancet Neurol 2016 Dec 21;15(13):1326-1335. Epub 2016 Oct 21.

Dementia Research Centre, Department of Neurodegenerative Disease, University College London Institute of Neurology, London, UK.

Background: The causes of phenotypic heterogeneity in familial Alzheimer's disease with autosomal dominant inheritance are not well understood. We aimed to characterise clinical phenotypes and genetic associations with APP and PSEN1 mutations in symptomatic autosomal dominant familial Alzheimer's disease (ADAD).

Methods: We retrospectively analysed genotypic and phenotypic data (age at symptom onset, initial cognitive or behavioural symptoms, and presence of myoclonus, seizures, pyramidal signs, extrapyramidal signs, and cerebellar signs) from all individuals with ADAD due to APP or PSEN1 mutations seen at the Dementia Research Centre in London, UK. We examined the frequency of presenting symptoms and additional neurological features, investigated associations with age at symptom onset, APOE genotype, and mutation position, and explored phenotypic differences between APP and PSEN1 mutation carriers. The proportion of individuals presenting with various symptoms was analysed with descriptive statistics, stratified by mutation type.

Findings: Between July 1, 1987, and Oct 31, 2015, age at onset was recorded for 213 patients (168 with PSEN1 mutations and 45 with APP mutations), with detailed history and neurological examination findings available for 121 (85 with PSEN1 mutations and 36 with APP mutations). We identified 38 different PSEN1 mutations (four novel) and six APP mutations (one novel). Age at onset differed by mutation, with a younger onset for individuals with PSEN1 mutations than for those with APP mutations (mean age 43·6 years [SD 7·2] vs 50·4 years [SD 5·2], respectively, p<0·0001); within the PSEN1 group, 72% of age at onset variance was explained by the specific mutation. A cluster of five mutations with particularly early onset (mean age at onset <40 years) involving PSEN1's first hydrophilic loop suggests critical functional importance of this region. 71 (84%) individuals with PSEN1 mutations and 35 (97%) with APP mutations presented with amnestic symptoms, making atypical cognitive presentations significantly more common in PSEN1 mutation carriers (n=14; p=0·037). Myoclonus and seizures were the most common additional neurological features; individuals with myoclonus (40 [47%] with PSEN1 mutations and 12 [33%] with APP mutations) were significantly more likely to develop seizures (p=0·001 for PSEN1; p=0·036 for APP), which affected around a quarter of the patients in each group (20 [24%] and nine [25%], respectively). A number of patients with PSEN1 mutations had pyramidal (21 [25%]), extrapyramidal (12 [14%]), or cerebellar (three [4%]) signs.

Interpretation: ADAD phenotypes are heterogeneous, with both age at onset and clinical features being influenced by mutation position as well as causative gene. This highlights the importance of considering genetic testing in young patients with dementia and additional neurological features in order to appropriately diagnose and treat their symptoms, and of examining different mutation types separately in future research.

Funding: Medical Research Council and National Institute for Health Research.
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http://dx.doi.org/10.1016/S1474-4422(16)30193-4DOI Listing
December 2016

Probing γ-secretase-substrate interactions at the single amino acid residue level.

EMBO J 2016 08 1;35(15):1597-9. Epub 2016 Jul 1.

VIB Center for the Biology of Disease, VIB-Leuven, Leuven, Belgium Center for Human Genetics, Universitaire Ziekenhuizen and LIND, KU Leuven, Leuven, Belgium Institute of Neurology, University College London, London, UK.

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http://dx.doi.org/10.15252/embj.201694978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4969572PMC
August 2016

Familial Alzheimer's Disease Mutations in Presenilin Generate Amyloidogenic Aβ Peptide Seeds.

Neuron 2016 04;90(2):410-6

VIB Center for the Biology of Disease, 3000 Leuven, Belgium; Center for Human Genetics, and Leuven Institute for Neurodegenerative Diseases (LIND), University of Leuven (KU Leuven), 3000 Leuven, Belgium; Institute of Neurology, University College London, London, WC1N 3BG, UK. Electronic address:

Recently it was proposed that the familial Alzheimer's disease (FAD) causing presenilin (PSEN) mutations PSEN1-L435F and PSEN1-C410Y do not support the generation of Aβ-peptides from the amyloid precursor protein (APP). This challenges the amyloid hypothesis and disagrees with previous work showing that PSEN1 FAD causing mutations generate invariably long Aβ and seed amyloid. We contrast here the proteolytic activities of these mutant PSEN alleles with the complete loss-of-function PSEN1-D257A allele. We find residual carboxy- and endo-peptidase γ-secretase activities, similar to the formerly characterized PSEN1-R278I. We conclude that the PSEN1-L435F and -C410Y mutations are extreme examples of the previously proposed "dysfunction" of γ-secretase that characterizes FAD-associated PSEN. This Matters Arising paper is in response to Xia et al. (2015), published in Neuron. See also the response by Xia et al. (2016), published in this issue.
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http://dx.doi.org/10.1016/j.neuron.2016.03.010DOI Listing
April 2016

Qualitative changes in human γ-secretase underlie familial Alzheimer's disease.

J Exp Med 2015 Nov 19;212(12):2003-13. Epub 2015 Oct 19.

VIB Center for the Biology of Disease, University of Leuven (KU Leuven), 3000 Leuven, Belgium Center for Human Genetics (CME) and Leuven Research Institute for Neuroscience and Disease (LIND), University of Leuven (KU Leuven), 3000 Leuven, Belgium

Presenilin (PSEN) pathogenic mutations cause familial Alzheimer's disease (AD [FAD]) in an autosomal-dominant manner. The extent to which the healthy and diseased alleles influence each other to cause neurodegeneration remains unclear. In this study, we assessed γ-secretase activity in brain samples from 15 nondemented subjects, 22 FAD patients harboring nine different mutations in PSEN1, and 11 sporadic AD (SAD) patients. FAD and control brain samples had similar overall γ-secretase activity levels, and therefore, loss of overall (endopeptidase) γ-secretase function cannot be an essential part of the pathogenic mechanism. In contrast, impaired carboxypeptidase-like activity (γ-secretase dysfunction) is a constant feature in all FAD brains. Significantly, we demonstrate that pharmacological activation of the carboxypeptidase-like γ-secretase activity with γ-secretase modulators alleviates the mutant PSEN pathogenic effects. Most SAD cases display normal endo- and carboxypeptidase-like γ-secretase activities. However and interestingly, a few SAD patient samples display γ-secretase dysfunction, suggesting that γ-secretase may play a role in some SAD cases. In conclusion, our study highlights qualitative shifts in amyloid-β (Aβ) profiles as the common denominator in FAD and supports a model in which the healthy allele contributes with normal Aβ products and the diseased allele generates longer aggregation-prone peptides that act as seeds inducing toxic amyloid conformations.
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http://dx.doi.org/10.1084/jem.20150892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4647268PMC
November 2015

Genetic determinants of white matter hyperintensities and amyloid angiopathy in familial Alzheimer's disease.

Neurobiol Aging 2015 Dec 4;36(12):3140-3151. Epub 2015 Sep 4.

Dementia Research Centre, Department of Neurodegenerative Diseases, UCL Institute of Neurology, London, UK.

Familial Alzheimer's disease (FAD) treatment trials raise interest in the variable occurrence of cerebral amyloid angiopathy (CAA); an emerging important factor in amyloid-modifying therapy. Previous pathological studies reported particularly severe CAA with postcodon 200 PSEN1 mutations and amyloid beta coding domain APP mutations. As CAA may manifest as white matter hyperintensities (WMH) on magnetic resonance imaging, particularly posteriorly, we investigated WMH in 52 symptomatic FAD patients for associations with mutation position. WMH were visually rated in 39 PSEN1 (18 precodon 200); 13 APP mutation carriers and 25 healthy controls. Ten PSEN1 mutation carriers (5 precodon 200) had postmortem examination. Increased WMH were observed in the PSEN1 postcodon 200 group and in the single APP patient with an amyloid beta coding domain (p.Ala692Gly, Flemish) mutation. WMH burden on MRI correlated with severity of CAA and cotton wool plaques in several areas. The precodon 200 group had younger ages at onset, decreased axonal density and/or integrity, and a greater T-lymphocytic response in occipital deep white matter. Mutation site contributes to the phenotypic and pathological heterogeneity witnessed in FAD.
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http://dx.doi.org/10.1016/j.neurobiolaging.2015.08.026DOI Listing
December 2015

The dynamic conformational landscape of gamma-secretase.

J Cell Sci 2015 Feb;128(3):589-98

The structure and function of the gamma-secretase proteases are of great interest because of their crucial roles in cellular and disease processes. We established a novel purification protocol for the gamma-secretase complex that involves a conformation- and complex-specific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the gamma-secretase structure and its conformational variability. Under steady-state conditions, the complex adopts three major conformations, which differ in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate-binding sites by inhibitors differentially stabilizes subpopulations of particles with compact conformations, whereas a mutation linked to familial Alzheimer disease results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the csecretase complex as a dynamic population of interconverting conformations, involving rearrangements at the nanometer scale and a high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect amyloid beta (Abeta) generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely to be instrumental for highly allosteric behavior of the enzyme.
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http://dx.doi.org/10.1242/jcs.164384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311135PMC
February 2015

Learning by failing: ideas and concepts to tackle γ-secretases in Alzheimer's disease and beyond.

Annu Rev Pharmacol Toxicol 2015 1;55:419-37. Epub 2014 Oct 1.

VIB Center for the Biology of Disease, Vlaams Instituut voor Biotechnologie, BE-3000 Leuven, Belgium.

γ-Secretases are a group of widely expressed, intramembrane-cleaving proteases involved in many physiological processes. Their clinical relevance comes from their involvement in Alzheimer's disease, cancer, and other disorders. A clinical trial with the wide-spectrum γ-secretase inhibitor semagacestat has, however, demonstrated that global inhibition of all γ-secretases causes serious toxicity. Evolving insights suggest that selective inhibition of one of these proteases, or more subtle modulation of γ-secretases by stimulating their carboxypeptidase-like activity but sparing their endopeptidase activity, are potentially highly interesting approaches. The rapidly growing knowledge of regulation, assembly, and specificity of these intriguing protein complexes and the potential advent of high-resolution structural information could dramatically change the perspective on safe and efficacious γ-secretase inhibition in various disorders.
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http://dx.doi.org/10.1146/annurev-pharmtox-010814-124309DOI Listing
September 2015

Signature amyloid β profiles are produced by different γ-secretase complexes.

J Biol Chem 2014 Feb 13;289(7):4346-55. Epub 2013 Dec 13.

From the Center for the Biology of Disease, Flemish Institute for Biology (VIB), 3000 Leuven, Belgium.

γ-Secretase complexes are involved in the generation of amyloid-β (Aβ) in the brain. Therefore, γ-secretase has been proposed as a potential therapeutic target in Alzheimer disease (AD). Targeting γ-secretase activity in AD requires the pharmacological dissociation of the processing of physiological relevant substrates and the generation of "toxic" Aβ. Previous reports suggest the differential targeting of γ-secretase complexes, based on their subunit composition, as a valid strategy. However, little is known about the biochemical properties of the different complexes, and key questions regarding their Aβ product profiles should be first addressed. Here, we expressed, purified, and analyzed, under the same conditions, the endopeptidase and carboxypeptidase-like activities of the four γ-secretase complexes present in humans. We find that the nature of the catalytic subunit in the complex affects both activities. Interestingly, PSEN2 complexes discriminate between the Aβ40 and Aβ38 production lines, indicating that Aβ generation in one or the other pathway can be dissociated. In contrast, the APH1 subunit mainly affects the carboxypeptidase-like activity, with APH1B complexes favoring the generation of longer Aβ peptides. In addition, we determined that expression of a single human γ-secretase complex in cell lines retains the intrinsic attributes of the protease while present in the membrane, providing validation for the in vitro studies. In conclusion, our data show that each γ-secretase complex produces a characteristic Aβ signature. The qualitative and quantitative differences between different γ-secretase complexes could be used to advance drug development in AD and other disorders.
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http://dx.doi.org/10.1074/jbc.M113.530907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924297PMC
February 2014

Dissecting γ-secretase function.

J Neurochem 2013 Apr;125(1):1-3

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http://dx.doi.org/10.1111/jnc.12077DOI Listing
April 2013

LRRK2 controls an EndoA phosphorylation cycle in synaptic endocytosis.

Neuron 2012 Sep;75(6):1008-21

VIB Center for the Biology of Disease, 3000 Leuven, Belgium.

LRRK2 is a kinase mutated in Parkinson's disease, but how the protein affects synaptic function remains enigmatic. We identified LRRK2 as a critical regulator of EndophilinA. Using genetic and biochemical studies involving Lrrk loss-of-function mutants and Parkinson-related LRRK2(G2019S) gain-of-kinase function, we show that LRRK2 affects synaptic endocytosis by phosphorylating EndoA at S75, a residue in the BAR domain. We show that LRRK2-mediated EndoA phosphorylation has profound effects on EndoA-dependent membrane tubulation and membrane association in vitro and in vivo and on synaptic vesicle endocytosis at Drosophila neuromuscular junctions in vivo. Our work uncovers a regulatory mechanism that indicates that reduced LRRK2 kinase activity facilitates EndoA membrane association, while increased kinase activity inhibits membrane association. Consequently, both too much and too little LRRK2-dependent EndoA phosphorylation impedes synaptic endocytosis, and we propose a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses.
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http://dx.doi.org/10.1016/j.neuron.2012.08.022DOI Listing
September 2012

The mechanism of γ-Secretase dysfunction in familial Alzheimer disease.

EMBO J 2012 May 13;31(10):2261-74. Epub 2012 Apr 13.

VIB Center for the Biology of Disease, Leuven, Belgium.

The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid β (Aβ)42 relative to Aβ40 by an unknown, possibly gain-of-toxic-function, mechanism. However, many PSEN mutations paradoxically impair γ-secretase and 'loss-of-function' mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aβ generation via three different mechanisms, resulting in qualitative changes in the Aβ profiles, which are not limited to Aβ42. Loss of ɛ-cleavage function is not generally observed among FAD mutants. On the other hand, γ-secretase inhibitors used in the clinic appear to block the initial ɛ-cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase-like (γ) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the Aβ products, and suggest fundamental improvements for current drug development efforts.
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http://dx.doi.org/10.1038/emboj.2012.79DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364747PMC
May 2012

Modification of γ-secretase by nitrosative stress links neuronal ageing to sporadic Alzheimer's disease.

EMBO Mol Med 2012 Jul 2;4(7):660-73. Epub 2012 May 2.

VIB Center for Biology of Disease - VIB, Leuven, Belgium.

Inherited familial Alzheimer's disease (AD) is characterized by small increases in the ratio of Aβ42 versus Aβ40 peptide which is thought to drive the amyloid plaque formation in the brain of these patients. Little is known however whether ageing, the major risk factor for sporadic AD, affects amyloid beta-peptide (Aβ) generation as well. Here we demonstrate that the secretion of Aβ is enhanced in an in vitro model of neuronal ageing, correlating with an increase in γ-secretase complex formation. Moreover we found that peroxynitrite (ONOO(-)), produced by the reaction of superoxide anion with nitric oxide, promoted the nitrotyrosination of presenilin 1 (PS1), the catalytic subunit of γ-secretase. This was associated with an increased association of the two PS1 fragments, PS1-CTF and PS1-NTF, which constitute the active catalytic centre. Furthermore, we found that peroxynitrite shifted the production of Aβ towards Aβ(42) and increased the Aβ(42) /Aβ(40) ratio. Our work identifies nitrosative stress as a potential mechanistic link between ageing and AD.
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http://dx.doi.org/10.1002/emmm.201200243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402223PMC
July 2012

Functional and topological analysis of Pen-2, the fourth subunit of the gamma-secretase complex.

J Biol Chem 2011 Apr 4;286(14):12271-82. Epub 2011 Feb 4.

Laboratory for the Research of Neurodegenerative Diseases, Center for Human Genetics, KULeuven, Leuven, Belgium.

The γ-secretase complex is a member of the family of intramembrane cleaving proteases, involved in the generation of the Aβ peptides in Alzheimer disease. One of the four subunits of the complex, presenilin, harbors the catalytic site, although the role of the other three subunits is less well understood. Here, we studied the role of the smallest subunit, Pen-2, in vivo and in vitro. We found a profound Notch-deficiency phenotype in Pen-2-/- embryos confirming the essential role of Pen-2 in the γ-secretase complex. We used Pen-2-/- fibroblasts to investigate the structure-function relation of Pen-2 by the scanning cysteine accessibility method. We showed that glycine 22 and proline 27 in hydrophobic domain 1 of Pen-2 are essential for complex formation and stability of γ-secretase. We also demonstrated that hydrophobic domain 1 and the loop domain of Pen-2 are located in a water-containing cavity and are in short proximity to the presenilin C-terminal fragment. We finally demonstrated the essential role of Pen-2 for the proteolytic activity of the complex. Our study supports the hypothesis that Pen-2 is more than a structural component of the γ-secretase complex and may contribute to the catalytic mechanism of the enzyme.
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http://dx.doi.org/10.1074/jbc.M110.216978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3069430PMC
April 2011

Inhibition of beta-secretase in vivo via antibody binding to unique loops (D and F) of BACE1.

J Biol Chem 2011 Mar 5;286(10):8677-87. Epub 2011 Jan 5.

Department of Molecular and Developmental Genetics, VIB, 3000 Leuven, Belgium.

β-Secretase (BACE1) is an attractive drug target for Alzheimer disease. However, the design of clinical useful inhibitors targeting its active site has been extremely challenging. To identify alternative drug targeting sites we have generated a panel of BACE1 monoclonal antibodies (mAbs) that interfere with BACE1 activity in various assays and determined their binding epitopes. mAb 1A11 inhibited BACE1 in vitro using a large APP sequence based substrate (IC(50) ∼0.76 nm), in primary neurons (EC(50) ∼1.8 nm), and in mouse brain after stereotactic injection. Paradoxically, mAb 1A11 increased BACE1 activity in vitro when a short synthetic peptide was used as substrate, indicating that mAb 1A11 does not occupy the active-site. Epitope mapping revealed that mAb 1A11 binds to adjacent loops D and F, which together with nearby helix A, distinguishes BACE1 from other aspartyl proteases. Interestingly, mutagenesis of loop F and helix A decreased or increased BACE1 activity, identifying them as enzymatic regulatory elements and as potential alternative sites for inhibitor design. In contrast, mAb 5G7 was a potent BACE1 inhibitor in cell-free enzymatic assays (IC(50) ∼0.47 nm) but displayed no inhibitory effect in primary neurons. Its epitope, a surface helix 299-312, is inaccessible in membrane-anchored BACE1. Remarkably, mutagenesis of helix 299-312 strongly reduced BACE1 ectodomain shedding, suggesting that this helix plays a role in BACE1 cellular biology. In conclusion, this study generated highly selective and potent BACE1 inhibitory mAbs, which recognize unique structural and functional elements in BACE1, and uncovered interesting alternative sites on BACE1 that could become targets for drug development.
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http://dx.doi.org/10.1074/jbc.M110.194860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048749PMC
March 2011

Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity.

J Biol Chem 2008 Jul 23;283(29):20096-105. Epub 2008 May 23.

Center for Human Genetics, KULeuven, and Department for Molecular and Developmental Genetics, VIB4, Herestraat 49, Leuven, Belgium.

The gamma-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin has been proposed to operate as the substrate receptor of the complex with the glutamate 332 (Glu(333) in human) serving as the anionic binding site for the alpha-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu(332) is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu(332) could bind the alpha-amino-terminal group of substrates, we hypothesized, in analogy with M28-aminopeptidases, that other residues in the putative binding site of Nicastrin should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of APP and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolishes the anionic alpha-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased gamma-secretase complex levels, but the remaining complex appeared as active as the wild-type complex. We confirmed in two independent in vitro assays that the specific enzymatic activity of the E332A mutant was comparable with that of the wild-type complex. Thus, Glu(332) crucially affects complex maturation rather than substrate recognition. Moreover other Nicastrin mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally gamma-secretase activity. Consequently, these studies indicate that the main role of the Glu(332) is in the maturation and assembly of gamma-secretase rather than in the recognition of the substrates.
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http://dx.doi.org/10.1074/jbc.M803040200DOI Listing
July 2008