Prevention of the β-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures

<p>Abstract</p> <p>Background</p> <p>Inflammation may be involved in the pathogenesis of Alzheimer's disease (AD). There has been little success with anti-inflammatory drugs in AD, while the promise of anti-inflammatory treatment is more evident in experimental models. A new anti-inflammatory strategy requires a better understanding of molecular mechanisms. Among the plethora of signaling pathways activated by β-amyloid (Aβ) peptides, the nuclear factor-kappa B (NF-κB) pathway could be an interesting target. In virus-infected cells, double-stranded RNA-dependent protein kinase (PKR) controls the NF-κB signaling pathway. It is well-known that PKR is activated in AD. This led us to study the effect of a specific inhibitor of PKR on the Aβ42-induced inflammatory response in primary mixed murine co-cultures, allowing interactions between neurons, astrocytes and microglia.</p> <p>Methods</p> <p>Primary mixed murine co-cultures were prepared in three steps: a primary culture of astrocytes and microglia for 14 days, then a primary culture of neurons and astrocytes which were cultured with microglia purified from the first culture. Before exposure to Aβ neurotoxicity (72 h), co-cultures were treated with compound C16, a specific inhibitor of PKR. Levels of tumor necrosis factor-α (TNFα), interleukin (IL)-1β, and IL-6 were assessed by ELISA. Levels of P_T451-PKR and activation of IκB, NF-κB and caspase-3 were assessed by western blotting. Apoptosis was also followed using annexin V-FITC immunostaining kit. Subcellular distribution of P_T451-PKR was assessed by confocal immunofluorescence and morphological structure of cells by scanning electron microscopy. Data were analysed using one-way ANOVA followed by a Newman-Keuls' post hoc test</p> <p>Results</p> <p>In these co-cultures, PKR inhibition prevented Aβ42-induced activation of IκB and NF-κB, strongly decreased production and release of tumor necrosis factor (TNFα) and interleukin (IL)-1β, and limited apoptosis.</p> <p>Conclusion</p> <p>In spite of the complexity of the innate immune response, PKR inhibition could be an interesting anti-inflammatory strategy in AD.</p

Towards precision medicine:What are the stratification hypotheses to identify homogeneous inflammatory subgroups

Diverse lines of research testify a link, presumably causal, between immune dysregulation and the development, course and clinical outcome of psychiatric disorders. However, there is a large heterogeneity among the patients? individual immune profile and this heterogeneity prevents the development of precise diagnostic tools and the identification of therapeutic targets. The aim of this review was to delineate possible subgroups of patients on the basis of clinical dimensions, investigating whether they could lead to particular immune signatures and tailored treatments. We discuss six clinical entry points; genetic liability to immune dysregula-tion, childhood maltreatment, metabolic syndrome, cognitive dysfunction, negative symptoms and treatment resistance. We describe the associated immune signature and outline the effects of anti-inflammatory drugs so far. Finally, we discuss advantages of this approach, challenges and future research directions. (c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field