TG2 promotes amyloid beta aggregates: Impact on ER-mitochondria crosstalk, calcium homeostasis and synaptic function in Alzheimer's disease.

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by cognitive impairment that increasingly affects the elderly. AD's main features have been related to cellular and molecular events, including the aberrant aggregation of the amyloid beta peptide (Aß), Ca2+ dyshomeostasis, and increased mitochondria-associated membranes (MAMs). Transglutaminase type 2 (TG2) is a ubiquitous enzyme whose primary role is the Ca2+-dependent proteins transamidation, including the Aß peptide. TG2 activity has been closely related to cellular damage and death. We detected increased TG2 levels in neuronal cells treated with Aß oligomers (AßOs) and hippocampal slices from J20 mice using cellular and molecular approaches. In this work, we characterized the capacity of TG2 to interact and promote Aß toxic aggregates (AßTG2). AßTG2 induced an acute increase in intracellular Ca2+, miniature currents, and hiperexcitability, consistent with an increased mitochondrial Ca2+ overload, IP3R-VDAC tethering, and mitochondria-endoplasmic reticulum contacts (MERCs). AßTG2 also decreased neuronal viability and excitatory postsynaptic currents, reinforcing the idea of synaptic failure associated with MAMs dysregulation mediated by TG2. Z-DON treatment, TG2 inhibitor, reduced calcium overload, mitochondrial membrane potential loss, and synaptic failure, indicating an involvement of TG2 in a toxic cycle which increases Aß aggregation, Ca2+ overload, and MAMs upregulation. These data provide novel information regarding the role TG2 plays in synaptic function and contribute additional evidence to support the further development of TG2 inhibitors as a disease-modifying strategy for AD.

Increased P2×2 receptors induced by amyloid-ß peptide participates in the neurotoxicity in alzheimer's disease.

Amyloid beta peptide (Aß) is tightly associated with the physiopathology of Alzheimer's Disease (AD) as one of the most important factors in the evolution of the pathology. In this context, we previously reported that Aß increases the expression of ionotropic purinergic receptor 2 (P2×2R). However, its role on the cellular and molecular Aß toxicity is unknown, especially in human brain of AD patients. Using cellular and molecular approaches in hippocampal neurons, PC12 cells, and human brain samples of patients with AD, we evaluated the participation of P2×2R in the physiopathology of AD. Here, we reported that Aß oligomers (Aßo) increased P2×2 levels in mice hippocampal neurons, and that this receptor increases at late Braak stages of AD patients. Aßo also increases the colocalization of APP with Rab5, an early endosomes marker, and decreased the nuclear/cytoplasmic ratio of Fe65 and PGC-1α immunoreactivity. The overexpression in PC12 cells of P2×2a, but not P2×2b, replicated these changes in Fe65 and PGC-1α; however, both overexpressed isoforms increased levels of Aß. Taken together, these data suggest that P2×2 is upregulated in AD and it could be a key potentiator of the physiopathology of Aß. Our results point to a possible participation in a toxic cycle that increases Aß production, Ca2+ overload, and a decrease of PGC-1α. These novel findings put the P2×2R as a key novel pharmacological target to develop new therapeutic strategies to treat Alzheimer's Disease.

Changes in PGC-1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive impairment that increasingly afflicts the elderly population. Soluble oligomers (AßOs) has been implicated in AD pathogenesis: however, the molecular events underlying a role for Aß are not well understood. We studied the effects of AßOs on mitochondrial function and on key proteins that regulate mitochondrial dynamics and biogenesis in hippocampal neurons and PC-12 cells. We find that AßOs treatment caused a reduction in total Mfn1 after a 2 h exposure (42 ± 11%); while DRP1 increased at 1 and 2 h (205 ± 22% and 198 ± 27%, respectively), correlating to changes in mitochondrial morphology. We also observed that SIRT1 levels were reduced after acute and chronic AßOs treatment (68 ± 7% and 77 ± 6%, respectively); while PGC-1α levels were reduced with the same time treatments (68 ± 8% and 67 ± 7%, respectively). Interestingly, we found that chronic treatment with AßOs increased the levels of pSIRT1 (24 h: 157 ± 18%), and we observed changes in the PGC-1α and p-SIRT1 nucleus/cytosol ratio and SIRT1-PGC-1α interaction pattern after chronic exposure to AßOs. Our data suggest that AßOs induce important changes in the level and localization of mitochondrial proteins related with the loss of mitochondrial function that are mediated by a fast and sustained SIRT1/PGC-1α complex disruption promoting a "non-return point" to an irreversible synaptic failure and neuronal network disconnection.

17 Oxo Sparteine and Lupanine, Obtained from Cytisus scoparius, Exert a Neuroprotection against Soluble Oligomers of Amyloid-ß Toxicity by Nicotinic Acetylcholine Receptors.

Alzheimer's disease (AD) is a neurodegenerative pathology, which is characterized by progressive and irreversible cognitive impairment. Most of the neuronal perturbations described in AD can be associated with soluble amyloid- ß oligomers (SO-Aß). There is a large amount of evidence demonstrating the neuroprotective effect of Nicotine neurotransmission in AD, mainly through nicotinic acetylcholine receptor (nAChR) activation and antiapoptotic PI3K/Akt/Bcl-2 pathway signaling. Using HPLC and GC/MS, we isolated and characterized two alkaloids obtained from C. scoparius, Lupanine (Lup), and 17- oxo-sparteine (17- ox), and examined their neuroprotective properties in a cellular model of SO-Aß toxicity. Our results showed that Lup and 17- ox (both at 0.03µM) prevented SO-Aß-induced toxicity in PC12 cells (Lup: 64±7%; 17- ox: 57±6%). Similar results were seen in hippocampal neurons where these alkaloids prevented SO-Aß neurotoxicity (Lup: 57±2%; 17- ox: 52±3%) and increased the frequency of spontaneous calcium transients (Lup: 60±4%; 17- Ox: 40±3%), suggesting an enhancing effect on neural network activity and synaptic activity potentiation. All of the neuroprotective effects elicited by both alkaloids were completely blocked by α-bungarotoxin. Additionally, we observed that the presence of both Lup and 17- ox increased Akt phosphorylation levels (52±4% and 35±7%, respectively) in cells treated with SO-Aß (3 h). Taken together, our results suggest that the activation of nAChR by Lup and 17- ox induces neuroprotection in different cellular models, and appears to be an interesting target for the development of new pharmacological tools and strategies against AD.

P2X receptor overexpression induced by soluble oligomers of amyloid beta peptide potentiates synaptic failure and neuronal dyshomeostasis in cellular models of Alzheimer's disease.

The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aß). Because Aß increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aß and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca2+ microfluorimetry to assess the effects of chronic exposure (24 h) to Aß oligomers (0.5 µM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aß. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca2+ signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aß. Taken together, our findings suggest that Aß disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.