Improved ELISPOT protocol for monitoring Th1/Th17 T-cell response following T.gondii infection.

In the monitoring of human Toxoplasma gondii infection, it is crucial to confirm the development of a specific Th1/Th17 immune response memory. The use of a simple, specific, and sensitive assay to follow the T-cell activation is thus required. Current protocols are not always specific as stimulation with peptides is Human Leukocyte Antigen (HLA)-dependent, while stimulation with total-lysis antigens tends to stimulate seronegative donors resulting to false positives. Here, an improved ELISPOT protocol is reported, using peripheral blood mononuclear cells (PBMC) of T.gondii-infected donors, incubated with the inactivated parasite. The results showed that, contrary to standard protocols, a pre-incubation step at high cell density in presence of the inactivated parasite allowed a specific Th1/Th17 response with the secretion of IFN-γ, IL-2, IL-12 and IL-17 cytokines. This protocol allows to evaluate precisely the immune response after a T.gondii infection.

The Alzheimer's disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons.

Bridging Integrator 1 (BIN1) is the second most important Alzheimer's disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing on cerebral organoids generated from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of hiPSC-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs that affect biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to slow disease-onset and progression.

Integration of Microfluidic Devices with Microelectrode Arrays to Functionally Assay Amyloid-ß-Induced Synaptotoxicity.

Alzheimer's disease (AD) is a neurodegenerative disease and the most frequent cause of dementia. It is characterized by the accumulation in the brain of two pathological protein aggregates: amyloid-ß peptides (Aß) and abnormally phosphorylated tau. The progressive cognitive decline observed in patients strongly correlates with the synaptic loss. Many lines of evidence suggest that soluble forms of Aß accumulate into the brain where they cause synapse degeneration. Stopping their spreading and/or targeting the pathophysiological mechanisms leading to synaptic loss would logically be beneficial for the patients. However, we are still far from understanding these processes. Our objective was therefore to develop a versatile model to assay and study Aß-induced synaptotoxicity. We integrated a microfluidic device that physically isolates synapses from presynaptic and postsynaptic neurons with a microelectrode array. We seeded mouse primary cortical cells in the presynaptic and postsynaptic chambers. After functional synapses have formed in the synaptic chamber, we exposed them to concentrated conditioned media from cell lines overexpressing the wild-type or mutated amyloid precursor protein and thus secreting different levels of Aß. We recorded the neuronal activity before and after exposition to Aß and quantified Aß's effects on the connectivity between presynaptic and postsynaptic neurons. We observed that the application of Aß on the synapses for 48 h strongly decreased the interchamber connectivity without significantly affecting the neuronal activity in the presynaptic or postsynaptic chambers. Thus, through this model, we are able to functionally assay the impact of Aß peptides (or other molecules) on synaptic connectivity and to use the latter as a proxy to study Aß-induced synaptotoxicity. Moreover, since the presynaptic, postsynaptic, and synaptic chambers can be individually targeted, our assay provides a powerful tool to evaluate the involvement of candidate genes in synaptic vulnerability and/or test therapeutic strategies for AD.

Nasal vaccination of six squirrel monkeys (Saimiri sciureus): Improved immunization protocol against Toxoplasma gondii with a nanoparticle-born vaccine.

Toxoplasma gondii is an intracellular protozoon found worldwide, which completes its life cycle between felids (its definitive host) and other warm-blooded animals. While the infection rarely leads to severe complications in humans, many animal species are very susceptible to this infection, for example the squirrel monkey (Saimiri sciureus) which is the subject of this study. Toxoplasmosis is lethal for 80% of cases in this species, and fatal outbreaks are frequently reported in zoological parks. No efficient treatment exists, but a new vaccine prepared with maltodextrin nanoparticles containing killed T. gondii antigens has been tested recently in French zoos. The animals were immunized through heterologous administrations, with two nasal doses at one-month interval, followed by nasal/subcutaneous boosts thereafter. No death has been reported since the beginning of this vaccination campaign, but we felt the protocol could be simplified. Here, an improved and less-invasive immunization protocol was evaluated on 6 Saimiri sciureus in the French zoo La Palmyre. It consisted of two nasal administrations at one-month interval, followed by a nasal boost at 6 months. A specific memory T-cell immunity was observed by ELISPOT after two administrations in all the animals, without humoral responses. The results suggest that 2 nasal administrations induce a protective immune response against T. gondii infection and might be sufficient to induce a strong Tcell memory, further improving immunity.

Pyk2 overexpression in postsynaptic neurons blocks amyloid ß1-42-induced synaptotoxicity in microfluidic co-cultures.

Recent meta-analyses of genome-wide association studies identified a number of genetic risk factors of Alzheimer's disease; however, little is known about the mechanisms by which they contribute to the pathological process. As synapse loss is observed at the earliest stage of Alzheimer's disease, deciphering the impact of Alzheimer's risk genes on synapse formation and maintenance is of great interest. In this article, we report a microfluidic co-culture device that physically isolates synapses from pre- and postsynaptic neurons and chronically exposes them to toxic amyloid ß peptides secreted by model cell lines overexpressing wild-type or mutated (V717I) amyloid precursor protein. Co-culture with cells overexpressing mutated amyloid precursor protein exposed the synapses of primary hippocampal neurons to amyloid ß1-42 molecules at nanomolar concentrations and induced a significant decrease in synaptic connectivity, as evidenced by distance-based assignment of postsynaptic puncta to presynaptic puncta. Treating the cells with antibodies that target different forms of amyloid ß suggested that low molecular weight oligomers are the likely culprit. As proof of concept, we demonstrate that overexpression of protein tyrosine kinase 2 beta-an Alzheimer's disease genetic risk factor involved in synaptic plasticity and shown to decrease in Alzheimer's disease brains at gene expression and protein levels-selectively in postsynaptic neurons is protective against amyloid ß1-42-induced synaptotoxicity. In summary, our lab-on-a-chip device provides a physiologically relevant model of Alzheimer's disease-related synaptotoxicity, optimal for assessing the impact of risk genes in pre- and postsynaptic compartments.

Impact of coxsackievirus-B4E2 combined with a single low dose of streptozotocin on pancreas of outbred mice: investigation of viral load, pathology and inflammation.

Coxsackieviruses B (CV-B) belong to the EV-B species. CV-B and particularly CV-B4 are thought to be involved in the development of chronic diseases like type 1 diabetes (T1D). The mechanisms of the enteroviral pathogenesis of T1D are not well known, yet. The in vitro studies are rich with information but in vivo infection models are needed to investigate the impact of viruses onto organs. Our objective was to study the impact of CV-B4E2 combined with a single sub-diabetogenic dose of streptozotocin (STZ) on the pancreas of mice. The infection with CV-B4E2 of CD1 outbred mice treated with a sub-diabetogenic dose of STZ induced hyperglycemia and hypoinsulinemia. Along with the chemokine IP-10, viral RNA and infectious particles were detected in the pancreas. The pancreas of these animals was also marked with insulitis and other histological alterations. The model combining STZ and CV-B4E2 opens the door to new perspectives to better understand the interactions between virus and host, and the role of environmental factors capable, like STZ, to predispose the host to the diabetogenic effects of enteroviruses.

BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr348 phosphorylation.

The bridging integrator 1 gene (BIN1) is a major genetic risk factor for Alzheimer's disease (AD). In this report, we investigated how BIN1-dependent pathophysiological processes might be associated with Tau. We first generated a cohort of control and transgenic mice either overexpressing human MAPT (TgMAPT) or both human MAPT and BIN1 (TgMAPT;TgBIN1), which we followed-up from 3 to 15 months. In TgMAPT;TgBIN1 mice short-term memory deficits appeared earlier than in TgMAPT mice; however-unlike TgMAPT mice-TgMAPT;TgBIN1 mice did not exhibit any long-term or spatial memory deficits for at least 15 months. After killing the cohort at 18 months, immunohistochemistry revealed that BIN1 overexpression prevents both Tau mislocalization and somatic inclusion in the hippocampus, where an increase in BIN1-Tau interaction was also observed. We then sought mechanisms controlling the BIN1-Tau interaction. We developed a high-content screening approach to characterize modulators of the BIN1-Tau interaction in an agnostic way (1,126 compounds targeting multiple pathways), and we identified-among others-an inhibitor of calcineurin, a Ser/Thr phosphatase. We determined that calcineurin dephosphorylates BIN1 on a cyclin-dependent kinase phosphorylation site at T348, promoting the open conformation of the neuronal BIN1 isoform. Phosphorylation of this site increases the availability of the BIN1 SH3 domain for Tau interaction, as demonstrated by nuclear magnetic resonance experiments and in primary neurons. Finally, we observed that although the levels of the neuronal BIN1 isoform were unchanged in AD brains, phospho-BIN1(T348):BIN1 ratio was increased, suggesting a compensatory mechanism. In conclusion, our data support the idea that BIN1 modulates the AD risk through an intricate regulation of its interaction with Tau. Alteration in BIN1 expression or activity may disrupt this regulatory balance with Tau and have direct effects on learning and memory.