Ibuprofen Favors Binding of Amyloid-β Peptide to Its Depot,Serum Albumin

The deposition of amyloid-β peptide (Aβ) in the brain is a critical event in the progression of Alzheimer’s disease (AD). This Aβ deposition could be prevented by directed enhancement of Aβ binding to its natural depot, human serum albumin (HSA). Previously, we revealed that specific endogenous ligands of HSA improve its affinity to monomeric Aβ. We show here that an exogenous HSA ligand, ibuprofen (IBU), exerts the analogous effect. Plasmon resonance spectroscopy data evidence that a therapeutic IBU level increases HSA affinity to monomeric Aβ₄₀/Aβ₄₂ by a factor of 3–5. Using thioflavin T fluorescence assay and transmission electron microcopy, we show that IBU favors the suppression of Aβ₄₀ fibrillation by HSA. Molecular docking data indicate partial overlap between the IBU/Aβ₄₀-binding sites of HSA. The revealed enhancement of the HSA–Aβ interaction by IBU and the strengthened inhibition of Aβ fibrillation by HSA in the presence of IBU could contribute to the neuroprotective effects of the latter, previously observed in mouse and human studies of AD.     

Development of Alkaline Phosphatase-Fused Mouse Prion Protein and Its Application in Toxic Aβ Oligomer Detection

Amyloid β (Aβ) oligomers play a key role in the progression of Alzheimer’s disease (AD). Multiple forms of Aβ assemblies have been identified by in vitro and in vivo analyses; however, it is uncertain which oligomer is highly neurotoxic. Thus, understanding the pathogenesis of AD by detecting toxic Aβ oligomers is crucial. In this study, we report a fusion protein of cellular prion protein (PrPc) and alkaline phosphatase (ALP) from Escherichia coli as a sensing element for toxic Aβ oligomers. Since the N-terminus domain of PrPc (residue 23–111) derived from mice is known to bind to toxic Aβ oligomers in vitro, we genetically fused PrPc_23–111 to ALP. The developed fusion protein, PrP–ALP, retained both the binding ability of PrPc and enzymatic activity of ALP. We showed that PrP–ALP strongly bound to high molecular weight (HMW) oligomers but showed little or no affinity toward monomers. The observation that PrP–ALP neutralized the toxic effect of Aβ oligomers indicated an interaction between PrP–ALP and toxic HMW oligomers. Based on ALP activity, we succeeded in detecting Aβ oligomers. PrP–ALP may serve as a powerful tool for detecting toxic Aβ oligomers that may be related to AD progression.     

Design and Experimental Evaluation of a Peptide Antagonist against Amyloid β(1–42) Interactions with Calmodulin and Calbindin-D28k

Amyloid β_1–42 (Aβ(1–42)) oligomers have been linked to the pathogenesis of Alzheimer’s disease (AD). Intracellular calcium (Ca²⁺) homeostasis dysregulation with subsequent alterations of neuronal excitability has been proposed to mediate Aβ neurotoxicity in AD. The Ca²⁺ binding proteins calmodulin (CaM) and calbindin-D28k, whose expression levels are lowered in human AD brains, have relevant roles in neuronal survival and activity. In previous works, we have shown that CaM has a high affinity for Aβ(1–42) oligomers and extensively binds internalized Aβ(1–42) in neurons. In this work, we have designed a hydrophobic peptide of 10 amino acid residues: VFAFAMAFML (amidated-C-terminus amino acid) mimicking the interacting domain of CaM with Aβ (1–42), using a combined strategy based on the experimental results obtained for Aβ(1–42) binding to CaM and in silico docking analysis. The increase in the fluorescence intensity of Aβ(1–42) HiLyte^TM-Fluor555 has been used to monitor the kinetics of complex formation with CaM and with calbindin-D28k. The complexation between nanomolar concentrations of Aβ(1–42) and calbindin-D28k is also a novel finding reported in this work. We found that the synthetic peptide VFAFAMAFML (amidated-C-terminus amino acid) is a potent inhibitor of the formation of Aβ(1–42):CaM and of Aβ(1–42):calbindin-D28k complexes.     

The Effects of Aβ1-42 Binding to the SARS-CoV-2 Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer’s disease (AD), the major form of dementia, β-amyloid (Aβ) levels in the blood are increased; however, the impact of elevated Aβ levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aβ_1-42, but not Aβ_1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aβ_1-42. Furthermore, Aβ_1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aβ_1-42 show that the clearance of Aβ_1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aβ antibody. In conclusion, these findings suggest that the binding of Aβ_1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aβ_1-42 in the blood is beneficial to the fight against COVID-19 and AD.     

Structure of N-Terminal Sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-Peptide with Phospholipase A2 from Venom of Andaman Cobra Sub-Species Naja naja sagittifera at 2.0 ? Resolution

Alzheimer’s disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD’s neuropathology. Studies have implicated the varied role of phospholipase A₂ (PLA₂) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA₂s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer’s Aβ peptide in its complex with PLA₂. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA₂ purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA₂ involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA₂ has the therapeutic potential of decreasing the Aβ–Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD.     

The Function of Transthyretin Complexes with Metallothionein in Alzheimer’s Disease

Alzheimer’s disease (AD) is one of the most frequently diagnosed types of dementia in the elderly. An important pathological feature in AD is the aggregation and deposition of the β-amyloid (Aβ) in extracellular plaques. Transthyretin (TTR) can cleave Aβ, resulting in the formation of short peptides with less activity of amyloid plaques formation, as well as being able to degrade Aβ peptides that have already been aggregated. In the presence of TTR, Aβ aggregation decreases and toxicity of Aβ is abolished. This may prevent amyloidosis but the malfunction of this process leads to the development of AD. In the context of Aβplaque formation in AD, we discuss metallothionein (MT) interaction with TTR, the effects of which depend on the type of MT isoform. In the brains of patients with AD, the loss of MT-3 occurs. On the contrary, MT-1/2 level has been consistently reported to be increased. Through interaction with TTR, MT-2 reduces the ability of TTR to bind to Aβ, while MT-3 causes the opposite effect. It increases TTR-Aβ binding, providing inhibition of Aβ aggregation. The protective effect, assigned to MT-3 against the deposition of Aβ, relies also on this mechanism. Additionally, both Zn₇MT-2 and Zn₇MT-3, decrease Aβ neurotoxicity in cultured cortical neurons probably because of a metal swap between Zn₇MT and Cu(II)Aβ. Understanding the molecular mechanism of metals transfer between MT and other proteins as well as cognition of the significance of TTR interaction with different MT isoforms can help in AD treatment and prevention.     

Natural Products Targeting Amyloid Beta in Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by severe brain damage and dementia. There are currently few therapeutics to treat this disease, and they can only temporarily alleviate some of the symptoms. The pathogenesis of AD is mainly preceded by accumulation of abnormal amyloid beta (Aβ) aggregates, which are toxic to neurons. Therefore, modulation of the formation of these abnormal aggregates is strongly suggested as the most effective approach to treat AD. In particular, numerous studies on natural products associated with AD, aiming to downregulate Aβ peptides and suppress the formation of abnormal Aβ aggregates, thus reducing neural cell death, are being conducted. Generation of Aβ peptides can be prevented by targeting the secretases involved in Aβ-peptide formation (secretase-dependent). Additionally, blocking the intra- and intermolecular interactions of Aβ peptides can induce conformational changes in abnormal Aβ aggregates, whereby the toxicity can be ameliorated (structure-dependent). In this review, AD-associated natural products which can reduce the accumulation of Aβ peptides via secretase- or structure-dependent pathways, and the current clinical trial states of these products are discussed.     

Impact of Amyloid-β on Platelet Mitochondrial Function and Platelet–Mediated Amyloid Aggregation in Alzheimer’s Disease

Background: Alzheimer’s disease (AD) is characterized by an accumulation of amyloid β (Aβ) peptides in the brain and mitochondrial dysfunction. Platelet activation is enhanced in AD and platelets contribute to AD pathology by their ability to facilitate soluble Aβ to form Aβ aggregates. Thus, anti-platelet therapy reduces the formation of cerebral amyloid angiopathy in AD transgenic mice. Platelet mitochondrial dysfunction plays a regulatory role in thrombotic response, but its significance in AD is unknown and explored herein. Methods: The effects of Aβ-mediated mitochondrial dysfunction in platelets were investigated in vitro. Results: Aβ40 stimulation of human platelets led to elevated reactive oxygen species (ROS) and superoxide production, while reduced mitochondrial membrane potential and oxygen consumption rate. Enhanced mitochondrial dysfunction triggered platelet-mediated Aβ40 aggregate formation through GPVI-mediated ROS production, leading to enhanced integrin αII_bβ₃ activation during synergistic stimulation from ADP and Aβ40. Aβ40 aggregate formation of human and murine (APP23) platelets were comparable to controls and could be reduced by the antioxidant vitamin C. Conclusions: Mitochondrial dysfunction contributes to platelet-mediated Aβ aggregate formation and might be a promising target to limit platelet activation exaggerated pathological manifestations in AD.     

TGF-β1 Protection against Aβ1–42-Induced Neuroinflammation and Neurodegeneration in Rats

Transforming growth factor (TGF)-β1, a cytokine that can be expressed in the brain, is a key regulator of the brain’s responses to injury and inflammation. Alzheimer’s disease (AD), the most common neurodegenerative disorder, involves inflammatory processes in the brain in addition to the hallmarks, amyloid-β (Aβ) plaques and neurofibrillary tangles. Recently, we have shown that T-helper (Th) 17 cells, a subpopulation of CD4⁺ T-cells with high proinflammation, also participate in the brain inflammatory process of AD. However, it is poorly known whether TGF-β1 ameliorates the lymphocyte-mediated neuroinflammation and, thereby, alleviates neurodegeneration in AD. Herein, we administered TGF-β1 via the intracerebroventricle (ICV) in AD model rats, by Aβ_1–42 injection in both sides of the hippocampus, to show the neuroprotection of TGF-β1. The TGF-β1 administration after the Aβ_1–42 injection ameliorated cognitive deficit and neuronal loss and apoptosis, reduced amyloid precursor protein (APP) expression, elevated protein phosphatase (PP)2A expression, attenuated glial activation and alleviated the imbalance of the pro-inflammatory/anti-inflammatory responses of T-lymphocytes, compared to the Aβ_1–42 injection alone. These findings demonstrate that TGF-β1 provides protection against AD neurodegeneration and suggest that the TGF-β1 neuroprotection is implemented by the alleviation of glial and T-cell-mediated neuroinflammation.     

Characterization of a Mouse Model of Alzheimer’s Disease Expressing Aβ4-42 and Human Mutant Tau

The relationship between the two most prominent neuropathological hallmarks of Alzheimer’s Disease (AD), extracellular amyloid-β (Aβ) deposits and intracellular accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFT), remains at present not fully understood. A large body of evidence places Aβ upstream in the cascade of pathological events, triggering NFTs formation and the subsequent neuron loss. Extracellular Aβ deposits were indeed causative of an increased tau phosphorylation and accumulation in several transgenic models but the contribution of soluble Aβ peptides is still controversial. Among the different Aβ variants, the N-terminally truncated peptide Aβ_4–42 is among the most abundant. To understand whether soluble Aβ_4–42 peptides impact the onset or extent of tau pathology, we have crossed the homozygous Tg4–42 mouse model of AD, exclusively expressing Aβ_4–42 peptides, with the PS19 (P301S) tau transgenic model. Behavioral assessment showed that the resulting double-transgenic line presented a partial worsening of motor performance and spatial memory deficits in the aged group. While an increased loss of distal CA1 pyramidal neurons was detected in young mice, no significant alterations in hippocampal tau phosphorylation were observed in immunohistochemical analyses.     

Design and Synthesis of Ranitidine Analogs as Multi-Target Directed Ligands for the Treatment of Alzheimer’s Disease

The aggregation of amyloid β (Aβ) peptides and deposition of amyloid plaques are implicated in the pathogenesis of Alzheimer’s disease (AD). Therefore, blocking Aβ aggregation with small molecules has been proposed as one therapeutic approach for AD. In the present study, a series of ranitidine analogs containing cyclic imide isosteres were synthesized and their inhibitory activities toward Aβ aggregation were evaluated using in vitro thioflavin T assays. The structure–activity relationship revealed that the 1,8-naphthalimide moiety provided profound inhibition of Aβ aggregation and structural modifications on the other parts of the parent molecule (compound 6) maintained similar efficacy. Some of these ranitidine analogs also possessed potent inhibitory activities of acetylcholinesterase (AChE), which is another therapeutic target in AD. These ranitidine analogs, by addressing both Aβ aggregation and AChE, offer insight into the key chemical features of a new type of multi-target directed ligands for the pharmaceutical treatment of AD.     

Assessing the Effects of Acute Amyloid β Oligomer Exposure in the Rat

Alzheimer’s disease (AD) is the most common form of dementia, yet there are no therapeutic treatments that can either cure or delay its onset. Currently, the pathogenesis of AD is still uncertain, especially with respect to how the disease develops from a normal healthy brain. Amyloid β oligomers (AβO) are highly neurotoxic proteins and are considered potential initiators to the pathogenesis of AD. Rat brains were exposed to AβO via bilateral intracerebroventricular injections. Rats were then euthanized at either 1, 3, 7 or 21-days post surgery. Rat behavioural testing was performed using the Morris water maze and open field tests. Post-mortem brain tissue was immunolabelled for Aβ, microglia, and cholinergic neurons. Rats exposed to AβO showed deficits in spatial learning and anxiety-like behaviour. Acute positive staining for Aβ was only observed in the corpus callosum surrounding the lateral ventricles. AβO exposed rat brains also showed a delayed increase in activated microglia within the corpus callosum and a decreased number of cholinergic neurons within the basal forebrain. Acute exposure to AβO resulted in mild learning and memory impairments with co-concomitant white matter pathology within the corpus callosum and cholinergic cell loss within the basal forebrain. Results suggest that acute exposure to AβO in the rat may be a useful tool in assessing the early phases for the pathogenesis of AD.     

Oligomeric and Fibrillar Species of Aβ42 Diversely Affect Human Neural Stem Cells

Amyloid-β 42 peptide (Aβ_1-42 (Aβ42)) is well-known for its involvement in the development of Alzheimer’s disease (AD). Aβ42 accumulates and aggregates in fibers that precipitate in the form of plaques in the brain causing toxicity; however, like other forms of Aβ peptide, the role of these peptides remains unclear. Here we analyze and compare the effects of oligomeric and fibrillary Aβ42 peptide on the biology (cell death, proliferative rate, and cell fate specification) of differentiating human neural stem cells (hNS1 cell line). By using the hNS1 cells we found that, at high concentrations, oligomeric and fibrillary Aβ42 peptides provoke apoptotic cellular death and damage of DNA in these cells, but Aβ42 fibrils have the strongest effect. The data also show that both oligomeric and fibrillar Aβ42 peptides decrease cellular proliferation but Aβ42 oligomers have the greatest effect. Finally, both, oligomers and fibrils favor gliogenesis and neurogenesis in hNS1 cells, although, in this case, the effect is more prominent in oligomers. All together the findings of this study may contribute to a better understanding of the molecular mechanisms involved in the pathology of AD and to the development of human neural stem cell-based therapies for AD treatment.     

The Neurovascular Unit Dysfunction in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common neurodegenerative disease worldwide. Histopathologically, AD presents with two hallmarks: neurofibrillary tangles (NFTs), and aggregates of amyloid β peptide (Aβ) both in the brain parenchyma as neuritic plaques, and around blood vessels as cerebral amyloid angiopathy (CAA). According to the vascular hypothesis of AD, vascular risk factors can result in dysregulation of the neurovascular unit (NVU) and hypoxia. Hypoxia may reduce Aβ clearance from the brain and increase its production, leading to both parenchymal and vascular accumulation of Aβ. An increase in Aβ amplifies neuronal dysfunction, NFT formation, and accelerates neurodegeneration, resulting in dementia. In recent decades, therapeutic approaches have attempted to decrease the levels of abnormal Aβ or tau levels in the AD brain. However, several of these approaches have either been associated with an inappropriate immune response triggering inflammation, or have failed to improve cognition. Here, we review the pathogenesis and potential therapeutic targets associated with dysfunction of the NVU in AD.     

Neurogenesis Is Increased in Human Neural Stem Cells by Aβ40 Peptide

Amyloid-β 40 peptides [Aβ1-40 (Aβ40)] are present within amyloid plaques in the brains of patients with Alzheimer’s disease (AD). Even though Aβ peptides are considered neurotoxic, they can mediate many biological processes, both in adult brains and throughout brain development. However, the physiological function of these Aβ peptides remains poorly understood, and the existing data are sometimes controversial. Here, we analyze and compare the effects of monomeric Aβ40 on the biology of differentiating human neural stem cells (human NSCs). For that purpose, we used a model of human NSCs called hNS1. Our data demonstrated that Aβ40 at high concentrations provokes apoptotic cellular death and the damage of DNA in human NSCs while also increasing the proliferation and favors neurogenesis by raising the percentage of proliferating neuronal precursors. These effects can be mediated, at least in part, by β-catenin. These results provide evidence of how Aβ modulate/regulate human NSC proliferation and differentiation, suggesting Aβ40 may be a pro-neurogenic factor. Our data could contribute to a better understanding of the molecular mechanisms involved in AD pathology and to the development of human NSC-based therapies for AD treatment, since these results could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.     

Cytotoxic Aβ Protofilaments Are Generated in the Process of Aβ Fibril Disaggregation

Significant research on Alzheimer’s disease (AD) has demonstrated that amyloid β (Aβ) oligomers are toxic molecules against neural cells. Thus, determining the generation mechanism of toxic Aβ oligomers is crucial for understanding AD pathogenesis. Aβ fibrils were reported to be disaggregated by treatment with small compounds, such as epigallocatechin gallate (EGCG) and dopamine (DA), and a loss of fibril shape and decrease in cytotoxicity were observed. However, the characteristics of intermediate products during the fibril disaggregation process are poorly understood. In this study, we found that cytotoxic Aβ aggregates are generated during a moderate disaggregation process of Aβ fibrils. A cytotoxicity assay revealed that Aβ fibrils incubated with a low concentration of EGCG and DA showed higher cytotoxicity than Aβ fibrils alone. Atomic force microscopy imaging and circular dichroism spectrometry showed that short and narrow protofilaments, which were highly stable in the β-sheet structure, were abundant in these moderately disaggregated samples. These results indicate that toxic Aβ protofilaments are generated during disaggregation from amyloid fibrils, suggesting that disaggregation of Aβ fibrils by small compounds may be one of the possible mechanisms for the generation of toxic Aβ aggregates in the brain.     

Ionic Environment Affects Biomolecular Interactions of Amyloid-β: SPR Biosensor Study

In early stages of Alzheimer’s disease (AD), amyloid beta (Aβ) accumulates in the mitochondrial matrix and interacts with mitochondrial proteins, such as cyclophilin D (cypD) and 17β-hydroxysteroid dehydrogenase 10 (17β-HSD10). Multiple processes associated with AD such as increased production or oligomerization of Aβ affect these interactions and disbalance the equilibrium between the biomolecules, which contributes to mitochondrial dysfunction. Here, we investigate the effect of the ionic environment on the interactions of Aβ (Aβ_1–40, Aβ_1–42) with cypD and 17β-HSD10 using a surface plasmon resonance (SPR) biosensor. We show that changes in concentrations of K⁺ and Mg²⁺ significantly affect the interactions and may increase the binding efficiency between the biomolecules by up to 35% and 65% for the interactions with Aβ_1–40 and Aβ_1–42, respectively, in comparison with the physiological state. We also demonstrate that while the binding of Aβ_1–40 to cypD and 17β-HSD10 takes place preferentially around the physiological concentrations of ions, decreased concentrations of K⁺ and increased concentrations of Mg²⁺ promote the interaction of both mitochondrial proteins with Aβ_1–42. These results suggest that the ionic environment represents an important factor that should be considered in the investigation of biomolecular interactions taking place in the mitochondrial matrix under physiological as well as AD-associated conditions.     

Impact of Two Neuronal Sigma-1 Receptor Modulators,PRE084 and DMT,on Neurogenesis and Neuroinflammation in an Aβ1–42-Injected,Wild-Type Mouse Model of AD

Alzheimer’s disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma-1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid-β-(Aβ-) induced neurotoxicity on the endoplasmic reticulum stress-associated pathways. Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aβ-induced in vivo mouse model characterizing neurogenic and anti-neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5-HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aβ_1–42-induced astrogliosis, but neither had remarkable effects on microglial activation. In summary, the highly selective S1R agonist PRE084 may be a promising therapeutic agent for AD. Further studies are required to clarify the multifaceted neurogenic and anti-neuroinflammatory roles of these agonists.     

Binding of Amyloid β(1–42)-Calmodulin Complexes to Plasma Membrane Lipid Rafts in Cerebellar Granule Neurons Alters Resting Cytosolic Calcium Homeostasis

Lipid rafts are a primary target in studies of amyloid β (Aβ) cytotoxicity in neurons. Exogenous Aβ peptides bind to lipid rafts, which in turn play a key role in Aβ uptake, leading to the formation of neurotoxic intracellular Aβ aggregates. On the other hand, dysregulation of intracellular calcium homeostasis in neurons has been observed in Alzheimer’s disease (AD). In a previous work, we showed that Aβ(1–42), the prevalent Aβ peptide found in the amyloid plaques of AD patients, binds with high affinity to purified calmodulin (CaM), with a dissociation constant ≈1 nM. In this work, to experimentally assess the Aβ(1–42) binding capacity to intracellular CaM, we used primary cultures of mature cerebellar granule neurons (CGN) as a neuronal model. Our results showed a large complexation of submicromolar concentrations of Aβ(1–42) dimers by CaM in CGN, up to 120 ± 13 picomoles of Aβ(1–42) /2.5 × 10⁶ cells. Using fluorescence microscopy imaging, we showed an extensive co-localization of CaM and Aβ(1–42) in lipid rafts in CGN stained with up to 100 picomoles of Aβ(1–42)-HiLyteTM-Fluor555 monomers. Intracellular Aβ(1–42) concentration in this range was achieved by 2 h incubation of CGN with 2 μM Aβ(1–42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 mM potassium medium. We conclude that the primary cause of the resting cytosolic calcium decrease is the inhibition of L-type calcium channels of CGN by Aβ(1–42) dimers, whose activity is inhibited by CaM:Aβ(1–42) complexes bound to lipid rafts.