Ana Maria Mata

The transformation of astrocytes into neurotoxic reactive astrocytes, classified as A1, by inflammatory cytokines, and their link to brain damage and neurodegenerative diseases has been widely documented. However, the roles of two biomarkers of Alzheimer's disease (AD), amyloid β‐peptide (Aβ) and tau, and that of calcium pumps which are involved in the fine‐tuning of calcium homeostasis, are poorly understood in astrocytes. In this study, we showed that treating astrocytoma U‐251 cells with a cocktail of cytokines significantly increased plasma membrane Ca²⁺‐ATPase (PMCA) activity and expression levels of the four PMCA isoforms. Moreover, treatment of cells with Aβ1‐42 or tau induced a similar upregulation of PMCA activity and isoform expression levels as cytokines. These effects support the close association of Aβ and tau with inflammation. This study may help better understand the role of PMCA in promoting calcium extrusion from astrocytes transformed by AD markers.

Highly neurotoxic A1-reactive astrocytes have been associated with several human neurodegenerative diseases. Complement protein C3 expression is strongly upregulated in A1 astrocytes, and this protein has been shown to be a specific biomarker of these astrocytes. Several cytokines released in neurodegenerative diseases have been shown to upregulate the production of amyloid β protein precursor (APP) and neurotoxic amyloid β (Aβ) peptides in reactive astrocytes. Also, aberrant Ca²⁺ signals have been proposed as a hallmark of astrocyte functional remodeling in Alzheimer’s disease mouse models. In this work, we induced the generation of A1-like reactive astrocytes after the co-treatment of U251 human astroglioma cells with a cocktail of the cytokines TNF-α, IL1-α and C1q. These A1-like astrocytes show increased production of APP and Aβ peptides compared to untreated U251 cells. Additionally, A1-like astrocytes show a (75 ± 10)% decrease in the Ca²⁺ stored in the endoplasmic reticulum (ER), (85 ± 10)% attenuation of Ca²⁺ entry after complete Ca²⁺ depletion of the ER, and three-fold upregulation of plasma membrane calcium pump expression, with respect to non-treated Control astrocytes. These altered intracellular Ca²⁺ dynamics allow A1-like astrocytes to efficiently counterbalance the enhanced release of Ca²⁺ from the ER, preventing a rise in the resting cytosolic Ca²⁺ concentration.

Dysregulation in calcium signaling pathways plays a major role in the initiation of Alzheimer’s disease (AD) pathogenesis. Accumulative experimental evidence obtained with cellular and animal models, as well as with AD brain samples, points out the high cytotoxicity of soluble small oligomeric forms of amyloid-β peptides (Aβ) in AD. In recent works, we have proposed that Aβ-calmodulin (CaM) complexation may play a major role in neuronal Ca²⁺ signaling, mediated by CaM-binding proteins (CaMBPs). STIM1, a recognized CaMBP, plays a key role in store-operated calcium entry (SOCE), and it has been shown that the SOCE function is diminished in AD, resulting in the instability of dendric spines and enhanced amyloidogenesis. In this work, we show that 2 and 5 h of incubation with 2 μM Aβ(1-42) oligomers of the immortalized mouse hippocampal cell line HT-22 leads to the internalization of 62 ± 11 nM and 135 ± 15 nM of Aβ(1-42), respectively. Internalized Aβ(1-42) oligomers colocalize with the endoplasmic reticulum (ER) and co-immunoprecipitated with STIM1, unveiling that this protein is a novel target of Aβ. Fluorescence resonance energy transfer measurements between STIM1 tagged with a green fluorescent protein (GFP) and Aβ(1-42)-HiLyte™-Fluor555 show that STIM1 can bind nanomolar concentrations of Aβ(1-42) oligomers at a site located close to the CaM-binding site in STIM1. Internalized Aβ(1-42) produced dysregulation of the SOCE in the HT-22 cells before a sustained alteration of cytosolic Ca²⁺ homeostasis can be detected, and is elicited by only 2 h of incubation with 2 μM Aβ(1-42) oligomers. We conclude that Aβ(1-42)-induced SOCE dysregulation in HT-22 cells is caused by the inhibitory modulation of STIM1, and the partial activation of ER Ca²⁺-leak channels.

Tissue degeneration is an event shared by many, if not all, age-related pathologies

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.

Plasma membrane calcium ATPases (PMCA) are key proteins in the maintenance of calcium (Ca²⁺) homeostasis. Dysregulation of PMCA function is associated with several human pathologies, including neurodegenerative diseases, and, therefore, these proteins are potential drug targets to counteract those diseases. Gold compounds, namely of Au(I), are well-known for their therapeutic use in rheumatoid arthritis and other diseases for centuries. Herein, we report the ability of dichloro(2-pyridinecarboxylate)gold(III) (1), chlorotrimethylphosphinegold(I) (2), 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidenegold(I) chloride (3), and chlorotriphenylphosphinegold(I) (4) compounds to interfere with the Ca²⁺-ATPase activity of pig brain purified PMCA and with membranes from SH-SY5Y neuroblastoma cell cultures. The Au(III) compound (1) inhibits PMCA activity with the IC₅₀ value of 4.9 µM, while Au(I) compounds (2, 3, and 4) inhibit the protein activity with IC₅₀ values of 2.8, 21, and 0.9 µM, respectively. Regarding the native substrate MgATP, gold compounds 1 and 4 showed a non-competitive type of inhibition, whereas compounds 2 and 3 showed a mixed type of inhibition. All gold complexes showed cytotoxic effects on human neuroblastoma SH-SY5Y cells, although compounds 1 and 3 were more cytotoxic than compounds 2 and 4. In summary, this work shows that both Au (I and III) compounds are high-affinity inhibitors of the Ca²⁺-ATPase activity in purified PMCA fractions and in membranes from SH-SY5Y human neuroblastoma cells. Additionally, they exert strong cytotoxic effects.

Since dysregulation of intracellular calcium (Ca²⁺) levels is a common occurrence in neurodegenerative diseases, including Alzheimer’s disease (AD), the study of proteins that can correct neuronal Ca²⁺ dysregulation is of great interest. In previous work, we have shown that plasma membrane Ca²⁺-ATPase (PMCA), a high-affinity Ca²⁺ pump, is functionally impaired in AD and is inhibited by amyloid-β peptide (Aβ) and tau, two key components of pathological AD hallmarks. On the other hand, sorcin is a Ca²⁺-binding protein highly expressed in the brain, although its mechanism of action is far from being clear. Sorcin has been shown to interact with the intracellular sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), and other modulators of intracellular Ca²⁺ signaling, such as the ryanodine receptor or presenilin 2, which is closely associated with AD. The present work focuses on sorcin in search of new regulators of PMCA and antagonists of Aβ and tau toxicity. Results show sorcin as an activator of PMCA, which also prevents the inhibitory effects of Aβ and tau on the pump, and counteracts the neurotoxicity of Aβ and tau by interacting with them.

Intraneuronal amyloid β (Aβ) oligomer accumulation precedes the appearance of amyloid plaques or neurofibrillary tangles and is neurotoxic. In Alzheimer’s disease (AD)-affected brains, intraneuronal Aβ oligomers can derive from Aβ peptide production within the neuron and, also, from vicinal neurons or reactive glial cells. Calcium homeostasis dysregulation and neuronal excitability alterations are widely accepted to play a key role in Aβ neurotoxicity in AD. However, the identification of primary Aβ-target proteins, in which functional impairment initiating cytosolic calcium homeostasis dysregulation and the critical point of no return are still pending issues. The micromolar concentration of calmodulin (CaM) in neurons and its high affinity for neurotoxic Aβ peptides (dissociation constant ≈ 1 nM) highlight a novel function of CaM, i.e., the buffering of free Aβ concentrations in the low nanomolar range. In turn, the concentration of Aβ-CaM complexes within neurons will increase as a function of time after the induction of Aβ production, and free Aβ will rise sharply when accumulated Aβ exceeds all available CaM. Thus, Aβ-CaM complexation could also play a major role in neuronal calcium signaling mediated by calmodulin-binding proteins by Aβ; a point that has been overlooked until now. In this review, we address the implications of Aβ-CaM complexation in the formation of neurotoxic Aβ oligomers, in the alteration of intracellular calcium homeostasis induced by Aβ, and of dysregulation of the calcium-dependent neuronal activity and excitability induced by Aβ.