Human Albumin Impairs Amyloid β-peptide Fibrillation Through its C-terminus: From docking Modeling to Protection Against Neurotoxicity in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative process characterized by the accumulation of extracellular deposits of amyloid β-peptide (Aβ), which induces neuronal death. Monomeric Aβ is not toxic but tends to aggregate into β-sheets that are neurotoxic. Therefore to prevent or delay AD onset and progression one of the main therapeutic approaches would be to impair Aβ assembly into oligomers and fibrils and to promote disaggregation of the preformed aggregate. Albumin is the most abundant protein in the cerebrospinal fluid and it was reported to bind Aβ impeding its aggregation. In a previous work we identified a 35-residue sequence of clusterin, a well-known protein that binds Aβ, that is highly similar to the C-terminus (CTerm) of albumin. In this work, the docking experiments show that the average binding free energy of the CTerm-Aβ1–42 simulations was significantly lower than that of the clusterin-Aβ1–42 binding, highlighting the possibility that the CTerm retains albumin's binding properties. To validate this observation, we performed in vitro structural analysis of soluble and aggregated 1 μM Aβ1–42 incubated with 5 μM CTerm, equimolar to the albumin concentration in the CSF. Reversed-phase chromatography and electron microscopy analysis demonstrated a reduction of Aβ1–42 aggregates when the CTerm was present. Furthermore, we treated a human neuroblastoma cell line with soluble and aggregated Aβ1–42 incubated with CTerm obtaining a significant protection against Aβ-induced neurotoxicity. These in silico and in vitro data suggest that the albumin CTerm is able to impair Aβ aggregation and to promote disassemble of Aβ aggregates protecting neurons

Pathophysiological regulation of the production, aggregation and toxicity of the amyloid beta-peptide in Alzheimer's disease

Society increase in live expectancy will cause a rise in Alzheimer’s Disease (AD) diagnosis, thus, there is a need to further understand how it works. One of the main culprits of this disease amyloid-β peptide (Aβ), this protein has a physiological function as monomers. But, once starts to aggregate, Aβ becomes neurotoxic. In this thesis, I have demonstrated a novel physiological function of monomeric Aβ as inductor of insulin receptor signalling, while its oligomers block the same signalling. I have described a neurotoxic feedback loop between oligomeric Aβ and an increase in BACE1 presence. I have studied novel calcium regulators in the development of the disease. I showed a novel pathway that links Aβ as a blocker of LTP. Finally, I have characterised the region of interaction of albumin c-terminus with Aβ and its neuroprotective effect.L’increment en l’esperança de vida a la nostre societat generarà un augment en el nombre de persones diagnosticades amb la malaltia d’ Alzheimer. Per això, és necessari poder comprendre millor el seu funcionament. Un dels principals actors d’aquesta malaltia es la proteïna β- amiloide, aquesta proteïna en estat monomèric te funcions fisiològiques, però quan agrega es torna neurotòxica. En aquesta tesis he demostrat una nova funció fisiològica de l’Aβ com a inductor de la senyalització regulada pel receptor d’insulina, mentre que els oligòmers la bloquegen. He descrit un cercle viciós entre Aβ oligomerica i un increment en la presencia de BACE1. He estudiat el paper de nous reguladors de calci en el desenvolupament d’AD. He mostrat una nova relació entre Aβ i el bloqueig del LTP. Finalment he caracteritzat la regió d’interacció del c-terminus de l’albúmina i l’Aβ i el seu paper neuroprotector

Nigella and milk thistle seed oils: potential cytoprotective effects against 7β-hydroxycholesterol-induced toxicity on SH-SY5Y cells

Oxysterols are assumed to be the driving force behind numerous neurodegenerative diseases. In this work, we aimed to study the ability of 7β-hydroxycholesterol (7β-OHC) to trigger oxidative stress and cell death in human neuroblastoma cells (SH-SY5Y) then the capacity of Nigella sativa and Milk thistle seed oils (NSO and MTSO, respectively) to oppose 7β-OHC-induced side effects. The impact of 7β-OHC, associated or not with NSO or MTSO, was studied on different criteria: cell viability; redox status, and apoptosis. Oxidative stress was assessed through the intracellular reactive oxygen species (ROS) production, levels of enzymatic and non-enzymatic antioxidants, lipid, and protein oxidation products. Our results indicate that 7β-OHC (40 µg/mL) exhibit pr-oxidative and pro-apoptotic activities shown by a decrease of the antioxidant enzymatic activities and an increase of ROS production, lipid, and protein oxidation end products as well as nitrotyrosine formation and caspase 3 activation. However, under the pre-treatment with NSO, and especially with MTSO (100 µg/mL), a marked attenuation of oxidative damages was observed. Our study suggests harmful effects of 7β-OHC consisting of pro-oxidative, anti-proliferative, and pro-apoptotic activities that may contribute to neurodegeneration. NSO and especially MTSO showed potential cytoprotection against the cytotoxicity of 7β-OHC.This study was supported by the Ministry of Higher Education and Scientific Research (MHESR), Tunisia. This work was also supported by the Spanish Ministry of Science and Innovation and FEDER Funds through grants SAF2017-83372-R (FJM) and through the “María de Maeztu” Program for Units of Excellence in R&D from Spain (award CEX2018-000792-M)

Amyloid β-Peptide Causes the Permanent Activation of CaMKIIα through Its Oxidation

Alzheimer’s disease (AD) is characterised by the presence of extracellular amyloid plaques in the brain. They are composed of aggregated amyloid beta-peptide (Aβ) misfolded into beta-sheets which are the cause of the AD memory impairment and dementia. Memory depends on the hippocampal formation and maintenance of synapses by long-term potentiation (LTP), whose main steps are the activation of NMDA receptors, the phosphorylation of CaMKIIα and the nuclear translocation of the transcription factor CREB. It is known that Aβ oligomers (oAβ) induce synaptic loss and impair the formation of new synapses. Here, we have studied the effects of oAβ on CaMKIIα. We found that oAβ produce reactive oxygen species (ROS), that induce CaMKIIα oxidation in human neuroblastoma cells as we assayed by western blot and immunofluorescence. Moreover, this oxidized isoform is significantly present in brain samples from AD patients. We found that the oxidized CaMKIIα is active independently of the binding to calcium/calmodulin, and that CaMKIIα phosphorylation is mutually exclusive with CaMKIIα oxidation as revealed by immunoprecipitation and western blot. An in silico modelling of the enzyme was also performed to demonstrate that oxidation induces an activated state of CaMKIIα. In brains from AD transgenic models of mice and in primary cultures of murine hippocampal neurons, we demonstrated that the oxidation of CaMKIIα induces the phosphorylation of CREB and its translocation to the nucleus to promote the transcription of ARC and BDNF. Our data suggests that CaMKIIα oxidation would be a pro-survival mechanism that is triggered when a noxious stimulus challenges neurons as do oAβ

Mitostasis, calcium and free radicals in health, aging and neurodegeneration

Mitochondria play key roles in ATP supply, calcium homeostasis, redox balance control and apoptosis, which in neurons are fundamental for neurotransmission and to allow synaptic plasticity. Their functional integrity is maintained by mitostasis, a process that involves mitochondrial transport, anchoring, fusion and fission processes regulated by different signaling pathways but mainly by the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). PGC-1α also favors Ca2+ homeostasis, reduces oxidative stress, modulates inflammatory processes and mobilizes mitochondria to where they are needed. To achieve their functions, mitochondria are tightly connected to the endoplasmic reticulum (ER) through specialized structures of the ER termed mitochondria-associated membranes (MAMs), which facilitate the communication between these two organelles mainly to aim Ca2+ buffering. Alterations in mitochondrial activity enhance reactive oxygen species (ROS) production, disturbing the physiological metabolism and causing cell damage. Furthermore, cytosolic Ca2+ overload results in an increase in mitochondrial Ca2+, resulting in mitochondrial dysfunction and the induction of mitochondrial permeability transition pore (mPTP) opening, leading to mitochondrial swelling and cell death through apoptosis as demonstrated in several neuropathologies. In summary, mitochondrial homeostasis is critical to maintain neuronal function; in fact, their regulation aims to improve neuronal viability and to protect against aging and neurodegenerative diseases.This work was supported by the Spanish Ministry of Science and Innovation and FEDER Funds through grants SAF2017-83372-R (FJM); RTI2018-094809-B-I00 (JMFF); PID2019-106755RB-I00 (RV); and through the “María de Maeztu” Programme for Units of Excellence in R&D (award CEX2018-000792-M)

Amyloid β-peptide causes the permanent activation of CaMKIIα through its oxidation

Alzheimer's disease (AD) is characterised by the presence of extracellular amyloid plaques in the brain. They are composed of aggregated amyloid beta-peptide (Aβ) misfolded into beta-sheets which are the cause of the AD memory impairment and dementia. Memory depends on the hippocampal formation and maintenance of synapses by long-term potentiation (LTP), whose main steps are the activation of NMDA receptors, the phosphorylation of CaMKIIα and the nuclear translocation of the transcription factor CREB. It is known that Aβ oligomers (oAβ) induce synaptic loss and impair the formation of new synapses. Here, we have studied the effects of oAβ on CaMKIIα. We found that oAβ produce reactive oxygen species (ROS), that induce CaMKIIα oxidation in human neuroblastoma cells as we assayed by western blot and immunofluorescence. Moreover, this oxidized isoform is significantly present in brain samples from AD patients. We found that the oxidized CaMKIIα is active independently of the binding to calcium/calmodulin, and that CaMKIIα phosphorylation is mutually exclusive with CaMKIIα oxidation as revealed by immunoprecipitation and western blot. An in silico modelling of the enzyme was also performed to demonstrate that oxidation induces an activated state of CaMKIIα. In brains from AD transgenic models of mice and in primary cultures of murine hippocampal neurons, we demonstrated that the oxidation of CaMKIIα induces the phosphorylation of CREB and its translocation to the nucleus to promote the transcription of ARC and BDNF. Our data suggests that CaMKIIα oxidation would be a pro-survival mechanism that is triggered when a noxious stimulus challenges neurons as do oAβ.This work was supported by the Spanish Ministry of Science and Innovation and Agencia Estatal de Investigación plus FEDER Funds through grants PID2020-117691RB-I00/AEI/10.13039/501100011033 (F.J.M.), SAF2017-83372-R (F.J.M.), BIO 2020-113203RB (B.O.) and PID2021-123482OB-I00 (A.O.). This work was also funded by the “María de Maetzu Programme” for Units of Excellence in R&D and the MM-MELIS intercollaborative projects (award CEX2018-000792-M/IPEP-MM2020-5). Funding for this project was also from the Comisión Nacional de Investigación Científica y Tecnológica-Chile: Fondecyt 12011668 (to A.R.Á.) and Millennium Science Initiative Program—ICN09_016/ICN 2021_045 (to A.R.Á.)

Differential regulation of insulin signalling by monomeric and oligomeric amyloid beta-peptide

Alzheimer's disease and Type 2 diabetes are pathological processes associated to ageing. Moreover, there are evidences supporting a mechanistic link between Alzheimer's disease and insulin resistance (one of the first hallmarks of Type 2 diabetes). Regarding Alzheimer's disease, amyloid β-peptide aggregation into β-sheets is the main hallmark of Alzheimer's disease. At monomeric state, amyloid β-peptide is not toxic but its function in brain, if any, is unknown. Here we show, by in silico study, that monomeric amyloid β-peptide 1-40 shares the tertiary structure with insulin and is thereby able to bind and activate insulin receptor. We validated this prediction experimentally by treating human neuroblastoma cells with increasing concentrations of monomeric amyloid β-peptide 1-40. Our results confirm that monomeric amyloid β-peptide 1-40 activates insulin receptor autophosphorylation, triggering downstream enzyme phosphorylations and the glucose Transporter 4 translocation to the membrane. On the other hand, neuronal insulin resistance is known to be associated to Alzheimer's disease since early stages. We thus modelled the docking of oligomeric amyloid β-peptide 1-40 to insulin receptor. We found that oligomeric amyloid β-peptide 1-40 blocks insulin receptor, impairing its activation. It was confirmed in vitro by observing the lack of insulin receptor autophosphorylation, and also the impairment of insulin-induced intracellular enzyme activations and the glucose Transporter 4 translocation to the membrane. By biological system analysis, we have carried out a mathematical model recapitulating the process that turns amyloid β-peptide binding to insulin receptor from the physiological to the pathophysiological regime. Our results suggest that monomeric amyloid β-peptide 1-40 contributes to mimic insulin effects in the brain, which could be good when neurons have an extra requirement of energy beside the well-known protective effects on insulin intracellular signalling, while its accumulation and subsequent oligomerization blocks the insulin receptor producing insulin resistance and compromising neuronal metabolism and protective pathways.This work was supported by the Spanish Ministry of Science and Innovation and Agencia Estatal de Investigación plus European Regional Development Fund (FEDER Funds) through grants PID2020-117691RB-I00/AEI/10.13039/501100011033 (F.J.M.), SAF2017-83372-R (F.J.M.), BIO2017-85329-R (B.O.), PGC2018-101251-B-I00 (J.G.-O.) and RTI2018-094579-B-I00 (X.F.-B.). This work was also funded by the Spanish Institute of Health Carlos III by project reference AC20/00009 -FEDER/UE and European Research Era Net (ERANET) ERA-CVD_JTC2020-015 (J.G.-O.), by the ‘María de Maeztu Programme’ for Units of Excellence in Research and Development (R&D; award CEX2018-000792-M), Generalitat de Catalunya (Spain) through the grant 2017-SGR-908 (X.F.-B.) and Fundación QUAES through Cátedra QUAES-UPF de Biomedicina e Ingeniería Biomédica. The Instituto de Salud Global de Barcelona (ISGlobal) and Institute for Bioengineering of Catalonia (IBEC) are members of the Centres de Recerca de Catalunya (CERCA) Programme, Generalitat de Catalunya (Spain). We acknowledge support from the Spanish Ministry of Science, Innovation and Universities through the ‘Centro de Excelencia Severo Ochoa 2019–2023’ Programme (CEX2018-000806-S). This research is part of ISGlobal's Programme on the Molecular Mechanisms of Malaria, which is partially supported by the Fundación Ramón Areces. J.G.-O. also acknowledges support from the Institució Catalana de Recerca i Estudis Avançats (ICREA) Academia programme

Amyloid beta-peptide increases BACE1 translation through the phosphorylation of the eukaryotic initiation factor-2 α

Alzheimer's disease (AD) is tightly linked to oxidative stress since amyloid beta-peptide (Aβ) aggregates generate free radicals. Moreover, the aggregation of Aβ is increased by oxidative stress, and the neurotoxicity induced by the oligomers and fibrils is in part mediated by free radicals. Interestingly, it has been reported that oxidative stress can also induce BACE1 transcription and expression. BACE1 is the key enzyme in the cleavage of the amyloid precursor protein to produce Aβ, and the expression of this enzyme has been previously shown to be enhanced in the brains of Alzheimer's patients. Here, we have found that BACE1 expression is increased in the hippocampi from AD patients at both the early (Braak stage II) and late (Braak stage VI) stages of the disease as studied by immunohistochemistry and western blot. To address the role of Aβ and oxidative stress in the regulation of BACE1 expression, we have analyzed the effect of subtoxic concentrations of Aβ oligomers (0.25 μM) and H2O2 (10 mM) on a human neuroblastoma cell line. Firstly, our results show that Aβ oligomers and H2O2 induce an increase of BACE1 mRNA as we studied by qPCR. Regarding BACE1 translation, it is dependent on the phosphorylation of the eukaryotic initiation factor 2α (eIF2α), since BACE1 mRNA bears a 5'UTR that avoids its translation under basal conditions. BACE1 5'UTR contains four upstream initiating codons (uAUGs), and its translation is activated when eIF2α is phosphorylated. Consistently, we have obtained that Aβ oligomers and H2O2 increase the levels of BACE1 and p-eIF2α assayed by western blot and confocal microscopy. Our results suggest that Aβ oligomers increase BACE1 translation by phosphorylating eIF2α in a process that involves oxidative stress and conforms a pathophysiological loop, where the Aβ once aggregated favors its own production continuously by the increase in BACE1 expression as observed in AD patients.This research was funded by the Spanish Ministry of Economy and Business through the grant Plan Estatal SAF2017-83372-R and SAF2014-52228-R (FEDER funds/UE) to FJM and RV, Chilean Government through Fondecyt 11611065 and AFB170005 to AA and REDES 180084 to AA and FJM, and MDM-2014-0370 through the María de Maeztu Programme for Units of Excellence in R&D to Departament de Ciències Experimentals i de la Salut. Silvia Menéndez is supported by the Health Deparment of the Generalitat de Catalunya, Spain (PERIS SLT006/17/00040). This work was also supported by the Advanced Microscopy Facility UC

Human Albumin Impairs Amyloid β-peptide Fibrillation Through its C-terminus : From docking Modeling to Protection Against Neurotoxicity in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative process characterized by the accumulation of extracellular deposits of amyloid β-peptide (Aβ), which induces neuronal death. Monomeric Aβ is not toxic but tends to aggregate into β-sheets that are neurotoxic. Therefore to prevent or delay AD onset and progression one of the main therapeutic approaches would be to impair Aβ assembly into oligomers and fibrils and to promote disaggregation of the preformed aggregate. Albumin is the most abundant protein in the cerebrospinal fluid and it was reported to bind Aβ impeding its aggregation. In a previous work we identified a 35-residue sequence of clusterin, a well-known protein that binds Aβ, that is highly similar to the C-terminus (CTerm) of albumin. In this work, the docking experiments show that the average binding free energy of the CTerm-Aβ simulations was significantly lower than that of the clusterin-Aβ binding, highlighting the possibility that the CTerm retains albumin's binding properties. To validate this observation, we performed in vitro structural analysis of soluble and aggregated 1 μM Aβ incubated with 5 μM CTerm, equimolar to the albumin concentration in the CSF. Reversed-phase chromatography and electron microscopy analysis demonstrated a reduction of Aβ aggregates when the CTerm was present. Furthermore, we treated a human neuroblastoma cell line with soluble and aggregated Aβ incubated with CTerm obtaining a significant protection against Aβ-induced neurotoxicity. These in silico and in vitro data suggest that the albumin CTerm is able to impair Aβ aggregation and to promote disassemble of Aβ aggregates protecting neurons

A Genome-Wide Functional Screen Identifies Enhancer and Protective Genes for Amyloid Beta-Peptide Toxicity

Alzheimer’s disease (AD) is known to be caused by amyloid β-peptide (Aβ) misfolded into β-sheets, but this knowledge has not yet led to treatments to prevent AD. To identify novel molecular players in Aβ toxicity, we carried out a genome-wide screen in Saccharomyces cerevisiae, using a library of 5154 gene knock-out strains expressing Aβ1–42. We identified 81 mammalian orthologue genes that enhance Aβ1–42 toxicity, while 157 were protective. Next, we performed interactome and text-mining studies to increase the number of genes and to identify the main cellular functions affected by Aβ oligomers (oAβ). We found that the most affected cellular functions were calcium regulation, protein translation and mitochondrial activity. We focused on SURF4, a protein that regulates the store-operated calcium channel (SOCE). An in vitro analysis using human neuroblastoma cells showed that SURF4 silencing induced higher intracellular calcium levels, while its overexpression decreased calcium entry. Furthermore, SURF4 silencing produced a significant reduction in cell death when cells were challenged with oAβ1–42, whereas SURF4 overexpression induced Aβ1–42 cytotoxicity. In summary, we identified new enhancer and protective activities for Aβ toxicity and showed that SURF4 contributes to oAβ1–42 neurotoxicity by decreasing SOCE activity