Role of Calpain in synaptic potentiation : link with CaMKII and Ca²⁺ signaling

La potentialisation synaptique dans les neurones d'hippocampes repose sur l'activation du récepteur NMDA (NMDAR) et l'influx de Ca²⁺. Des changements dans le Ca²⁺ cytosolique sont détectés par des effecteurs tels que la calpaïne et la protéine kinase II Ca²⁺/calmoduline-dépendante (CaMKII), transformant ces informations en signaux qui induisent une potentialisation synaptique. Une fois activée par l'influx de Ca²⁺, la calpaïne clive de nombreuses protéines cytosoliques, récepteurs et protéines d'échafaudage, remodelant ainsi la structure synaptique, ainsi que l'activité et/ou la dynamique de nombreuses protéines. Le rôle de la calpaïne au cours du processus de plasticité synaptique a été documenté, mais le mécanisme moléculaire est loin d'être clair. Dans cette étude, nous avons examiné le lien possible entre la calpaïne et CaMKII dans la médiation de la potentialisation à long terme (LTP). Nous avons utilisé des inhibiteurs pharmacologiques de la calpaïne pour interférer avec son activation lors de la potentialisation synaptique induite chimiquement dans des cultures dissociées d'hippocampe de rat. Nous avons d'abord confirmé que l'activité de la calpaïne est essentielle pour l'induction de la LTP dans les cultures neuronales dissociées. Nous montrons que l'activité de la calpaïne est essentielle pour de nombreux processus moléculaires importants pour la LTP. L'inhibition de l'activité de la calpaïne a bloqué la phosphorylation de ERK et l'insertion des récepteurs synaptiques AMPA; deux processus régulés par CaMKII impliqués dans la potentialisation synaptique. De plus, nous montrons que la calpaïne est essentielle pour l'autophosphorylation de CaMKII en utilisant un anticorps contre pCaMKII (Thr286). En mesurant le temps de vie par fluorescence (FLIM) avec un capteur basé sur le transfert d'énergie par résonance de fluorescence (FRET) (Camui) de l'activation de CaMKII, nous montrons que l'inhibition de la calpaïne empêche le changement dépendant de l'activité de la conformation de l'holoenzyme CaMKII et donc l'activation de la kinase. Nous avons aussi utilisé l'imagerie time-lapse et avons découvert que la translocation CaMKII post-synaptique dépendante de l'activité est diminuée par les inhibiteurs de la calpaïne. De plus, nous avons mesuré les taux de diffusion de CaMKII par SPT-PALM en utilisant CaMKII-meos2 et les résultats indiquent que l'inhibition de la calpaïne empêche la diminution dépendante de l'activité de la mobilité de l'holoenzyme. Nos résultats montrent clairement que les inhibiteurs de la calpaïne affectent la dynamique de CaMKII. Cela suggère que la calpaïne affecte directement CaMKII ou agit en amont de CaMKII. En effectuant des expériences dans des cellules HEK qui n'ont pas de CaMKII endogène, nous avons démontré que la calpaïne n'affecte pas directement CaMKII. Nous avons émis l'hypothèse que la calpaïne joue un rôle dans le processus de plasticité en amont de CaMKII. Nous avons étudié l'influx Ca²⁺ dépendant de l'activité en utilisant l'imagerie GCaMP6 et nos résultats indiquent que l'activité de la calpaïne est essentielle pour cette l'augmentation de Ca²⁺. En disséquant davantage la voie de signalisation, utilisant différents protocoles de stimulation (dépolarisation synaptique ou globale), nous montrons que la calpaïne affecte l'afflux de Ca²⁺ dépendant de NMDA et non l'influx de Ca²⁺ dépendant de la dépolarisation. Ainsi, notre étude montre que la calpaïne joue un rôle essentiel dans la LTP d'une manière dépendante du NMDAR et que l'inhibition de la calpaïne interfère dans les premières étapes de la signalisation médiée par le Ca²⁺ conduisant à l'induction du LTP. En discutant de ces résultats, nous fournissons des résultats préliminaires qui peuvent nous éclairer au niveau de l'impact de l'inhibition pharmacologique de la calpaïne sur la fonction des récepteurs NMDA.Synaptic potentiation in hippocampal neurons relies on NMDA receptor (NMDAR) activation and Ca²⁺ influx. Changes in cytosolic Ca²⁺ are detected by effectors such as calpain and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), transforming this information into signals inducing synaptic potentiation. Once activated by Ca²⁺ influx, calpain cleaves many cytosolic proteins, receptors, and scaffolding proteins, thereby remodeling the synaptic structure, as well as the activity and/or dynamics of many proteins. The role of calpain during the synaptic plasticity process has been documented, but the molecular mechanism is far from clear. In this study, we examined the possible link between calpain and CaMKII in the mediation of Long Term Potentiation (LTP). We used pharmacological inhibitors of calpain to interfere with its activation during chemically induced synaptic potentiation in rat hippocampal dissociated cultures. We first confirmed that calpain activity is essential for LTP induction in dissociated neuronal cultures. We show that calpain activity is essential for many molecular processes important for LTP. Inhibition of calpain activity blocked ERK phosphorylation and insertion of synaptic AMPA receptors - two CaMKII-regulated processes involved in synaptic potentiation. Further, we show that calpain is essential for CaMKII autophosphorylation by using an antibody against pCaMKII (Thr286). By performing Fluorescence Lifetime Imaging (FLIM) with a fluorescence resonance energy transfer (FRET)-based sensor (Camui) of CaMKII activation, we show that calpain inhibition prevents activity-dependent change in the conformation of the CaMKII holoenzyme and thus the activation of the kinase. We further used time-lapse imaging and found that activity-dependent post-synaptic CaMKII translocation is decreased by calpain inhibitors. Furthermore, we measured diffusion rates of CaMKII by SPT-PALM using CaMKII-meos2 and the results indicate that calpain inhibition prevents the activity-dependent decrease in the mobility of the holoenzyme. Our results clearly show that calpain inhibitors affect CaMKII dynamics. This suggests that either calpain affects CaMKII directly or is upstream to CaMKII. By performing experiments in HEK cells that do not have endogenous CaMKII, we demonstrated that calpain does not affect CaMKII directly. We hypothesized that calpain plays a role in the plasticity process at an upstream level to CaMKII. We investigated activity-dependent Ca²⁺ influx using GCaMP6 imaging and our results indicate that calpain activity is essential for this increase in Ca²⁺. Further dissecting the pathway, using different stimulation protocols (synaptic or global depolarisation), we show that calpain affects NMDA-dependent Ca²⁺ influx and not the depolarisation dependent Ca²⁺ influx. Thus, our study shows that calpain plays an essential role in LTP in an NMDAR dependent manner and that inhibiting calpain interferes in the early steps of Ca²⁺- mediated signaling leading to LTP induction. In discussing these results, we provide preliminary results that may shed light on the impact of pharmacological inhibition of calpain on NMDA receptor function

Malaria in Pregnancy

Malaria infection during pregnancy is an important public health problem with substantial risks to both the mother and foetus. Pregnant women are the most vulnerable group of malaria‐associated morbidity and mortality. A pregnant woman has an increased risk (up to four times) of getting malaria and twice the chances of dying from malaria, compared to a non‐pregnant adult, becuase the immune system is partially suppressed during pregnancy. Malaria in pregnancy not only affects the mother but also has a dangerous sequel for the developing foetus, resulting in premature delivery or intrauterine growth retardation. Diagnosis of malaria in pregnancy remains a challenge due to the low parasite density and placental sequestration of Plasmodium falciparum. Thus, there is an urgent need for new diagnostic methods to detect malarial parasites in the pregnant women. Though antimalarial drugs are available, which can be safely given in the pregnancy, increasing drug resistance of malarial parasite may pose a big problem in the future. In this chapter, we review the burden of pregnancy‐associated malaria (PAM), its pathogenesis, diagnostic issues during pregnancy and recent guidelines for chemoprophylaxsis and treatment

Trichomoniasis and Lactoferrin: Future Prospects

Trichomonas vaginalis is a parasitic protozoan which infects the urogenital tract and requires iron as an essential nutrient. Iron is known to upregulate various adhesins required for cytoadherance and other factors involved in pathogenesis. At mucosal surfaces, iron is chelated by lactoferrin resulting in low levels of free iron. However, pathogens have evolved mechanisms for an increased uptake of iron. The present review highlights the role of iron in survival of Trichomonas during fluctuating concentrations of iron at mucosal surfaces during the menstrual cycle. Future prospects in terms of new drug and vaccine targets related to iron and its receptors have also been described

New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3'UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future

CPEB3a phosphorylation in cultured hippocampal neurons.

<p>Normalized ratio of phosphorylated CPEB3a (phospho-CPEB3a) fluorescence over total CPEB3a (CPEB3a-EGFP) fluorescence (n = 25–35, 3 experiments). (<b>A</b>) Immunofluorescence of neurons transfected with CPEB3a-mGFP subjected to a 40 mM KCl stimulation for 90 s, which leads to a marked increase in phospho-CPEB3a signal. One hour pretreatment with 10 μM KN93 or 10μM H89 in the stimulation medium reverts this increase. Co-transfection of the natural CaMKII inhibitor (CaMKIIN-HA) prevents the increase in CPEB3a phosphorylation. (<b>B</b>) Quantification of the ratio of phospho-CPEB3a over CPEB3a-mGFP signals in A, which shows a significant increase of CPEB3a phosphorylation following KCl stimulation only, which is not seen in neurons treated with KN93, H89, or those co-transfected with CaMKIIN-HA; n = 10–35, at least 3 experiments per condition. (<b>C</b>) Immunofluorescence of neurons transfected with CPEB3a-mGFP and subjected to either 5 min 50 μM FS stimulation or 1 h 300 nm OA stimulation, which both cause a robust increase in phospho-CPEB3a signal. Pretreatment with 10 μM H89 or 100 μM RP-cAMPS for 1 h partially reverts the effect of FS. (<b>A</b>, <b>C</b>) The scale bar represents 10 μm and applies to all photomicrographs. (<b>D</b>) Quantification of the ratio of phospho-CPEB3a over CPEB3a-mGFP signals in C, which shows a significant increase of CPEB3a phosphorylation following FS, OA and combined FS + OA treatments. Increase in phosphorylation induced by FS is partially reversed by cAMPS-Rp treatment; n = 22–59, 2–5 experiments per condition. (<b>A</b>, <b>B</b>, <b>C</b>, <b>D</b>) *p<0.05, Kruskal-Wallis followed by Tukey’s least-significant difference test.</p

Summary of mass spectrometric evidence for phosphorylation of CPEB3a by PKA, CaMKII and endogenous kinases present in HEK-293 cells.

<p>Summary of mass spectrometric evidence for phosphorylation of CPEB3a by PKA, CaMKII and endogenous kinases present in HEK-293 cells.</p

qRT-PCR analysis of CPEB3 isoforms of hippocampal mRNA after intraperitoneal kainate injection.

<p>The mRNA levels of isoforms with the B-region increased 30 min after kainate-induced <i>status epilepticus</i>, while the levels of the isoforms lacking the B-region remained unchanged. n = 4 animals = 8 hippocampi (per condition). *p<0.05, student’s t test.</p

Multiple sequence alignment of the regions flanking S419/S420 of CPEBs-2-4.

<p>Phosphorylation site was marked with a black asterisk. Residue conservation score was calculated with Jalview software according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150000#pone.0150000.ref061" target="_blank">61</a>]. Color intensity and the score (1–11) in the “conservation index” (below the alignment) reflects the conservation of physicochemical properties of amino acids in the particular column of the alignment. *: conserved column (score 11, highest); +: all the amino acid physical properties conserved (score 10).</p