Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotectio

Differential Contribution of L-, N-, and P/Q-type Calcium Channels to [Ca2+]i Changes Evoked by Kainate in Hippocampal Neurons

Abstract We investigated the contribution of L-, N- and P/Q-type Ca2+ channels to the [Ca2+]i changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca2+ imaging. Selective Ca2+ channel blockers, namely nitrendipine, ?-Conotoxin GVIA (?-GVIA) and ?-Agatoxin IVA (?-AgaIVA) were used. The [Ca2+]i changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but the N-methyl-d-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca2+ channel blocker caused differential inhibitory effects on [Ca2+]i responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above 90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by ?-GVIA or ?-AgaIVA. Thus, in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case of ?-GVIA or ?-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what concerns their L-, N- and P/Q- type Ca2+ channels activated by stimulation of the AMPA/kainate receptors

Microglia Dysfunction Caused by the Loss of Rhoa Disrupts Neuronal Physiology and Leads to Neurodegeneration

Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of ß-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aß oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.The authors acknowledge the support of the following i3S Scientific Platforms: Animal Facility, Translational Cytometry Unit (TraCy), BioSciences Screening (BS) and Advanced Light Microscopy (ALM), and members of the national infrastructure PPBI-Portuguese Platform of BioImaging (supported by POCI-01–0145-FEDER-022122). FCT Portugal (PTDC/MED-NEU/31318/2017-031318) supported work in the J.B.R. lab. FCT Portugal, PEst (UID/NEU/04539/2013), COMPETE-FEDER (POCI-01-0145-FEDER-007440), Centro 2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000008: BrainHealth 2020), and Strategic Project UIDB/04539/2020 and UIDP/04539/2020 (CIBB) supported work in the A.F.A. lab. C.C.P. and R.S. hold employment contracts financed by national funds through FCT (Fundação para a Ciência e a Tecnologia, I.P.) in the context of the program contract described in paragraphs 4, 5, and 6 of article 23 of law no. 57/2016, of August 29th, as amended by law no. 57/2017 of July 19th

Potencial de híbridos e populações segregantes de abóbora para teor de óleo nas sementes e plantas com crescimento do tipo moita

O objetivo deste estudo foi caracterizar e avaliar populações híbridas e segregantes, provenientes de cruzamentos entre acessos de Cucurbita moschata, com potencial oleaginoso, e cultivares que apresentam o gene para crescimento do tipo moita (Bush), visando à obtenção de genótipos com elevado teor de óleo nas sementes e com crescimento da planta do tipo moita. O delineamento experimental utilizado foi o de blocos casualizados, com três repetições. Cada repetição foi constituída por cinco plantas dos acessos BGH-7319 e BGH-7765, dos cultivares Piramoita e Tronco Verde e dos híbridos F1 (BGH-7319 x Piramoita, BGH-7319 x Tronco Verde, BGH-7765 x Tronco Verde), sendo consideradas as três plantas centrais das parcelas como úteis. Para cada uma das três populações F2 (‘População 1 F2’, ‘População 2 F2’, ‘População 3 F2’), 30 plantas foram avaliadas. Foram utilizados 28 descritores fenotípicos, sendo nove relativos à fase vegetativa das plantas, 12 referentes aos frutos e sete às sementes. Os dados foram submetidos à análise de variância e as médias dos tratamentos contrastadas com as médias das testemunhas, a 5% de probabilidade, pelo teste Dunnett. Na predição do potencial das populações para obtenção de linhagens superiores, foi utilizada a metodologia de Jinks & Pooni (1976). O híbrido BGH-7319 x Tronco Verde destaca-se para os caracteres massa de sementes por fruto, massa de cem sementes, comprimento, largura e espessura da semente, sendo considerada promissora quanto ao teor de óleo nas sementes. A ‘População 2 F2’ derivada do cruzamento entre os genitores BGH-7319 e Tronco Verde foi considerada a mais promissora para planta com crescimento do tipo moita.The objective of this study was to characterize and evaluate hybrid and segregating populations of crosses between accessions of Cucurbita moschata, with oil potential, and cultivars that possess the gene of dwarfism (bush), aiming at obtaining genotypes with high seed oil yield and reduced plant architecture. The experimental design was in randomized blocks with three replicates. Each replicate consisted of five plants of the BGH-7319 and BGH-7765 accessions, of the cultivars Piramoita and Tronco Verde and the F1 hybrids (BGH-7319 × Piramoita, BGH-7319 × Tronco Verde, BGH-7765 × Tronco Verde), considering the three central plants of the plots as useful. Thirty plants for each of the three F2 populations ('Population 1 F2', 'Population 2 F2', 'Population 3 F2') were evaluated. Twenty-eight phenological descriptors were applied, nine of which were related to the vegetative phase of the plants, 12 referring to the fruits, and seven to the seeds. The data were subjected to analysis of variance and the means of the treatments were compared with the means of the controls at 5% of probability by Dunnett’s test. In predicting the potential of populations to obtain superior strains, the methodology of Jinks & Pooni (1976) was used. The hybrid BGH-7319 × Tronco Verde stood out for the characteristics of seed mass per fruit, mass of 100 seeds, and seed length, width, and thickness, being promising for the oil content of the seeds. The 'Population 2 F2', derived from the crossbreeding between BGH-7319 and Tronco Verde was to the most promising for plant with shrub-type growth