Genetic Inactivation of Free Fatty Acid Receptor 3 Impedes Behavioral Deficits and Pathological Hallmarks in the APP Swe Alzheimer’s Disease Mouse Model

The free fatty acid FFA3 receptor (FFA3R) belongs to the superfamily of G-protein-coupled receptors (GPCRs). In the intestine and adipose tissue, it is involved in the regulation of energy metabolism but its function in the brain is unknown. We aimed, first, to investigate the expression of the receptor in the hippocampus of Alzheimer disease (AD) patients at different stages of the disease and, second, to assess whether genetic inactivation of the Ffar3 gene could affect the phenotypic features of the APPswe mouse model. The expression of transcripts for FFA receptors in post mortem human hippocampal samples and in the hippocampus of wild-type and transgenic mice was analyzed by RT-qPCR. We generated a double transgenic mouse, FFA3R-/-/APPswe, to perform cognition studies and to assess, by immunoblotting, Aβ and tau pathologies and the differential expression of synaptic plasticity-related proteins.For the first time, the occurrence of the FFA3R in the human hippocampus and its overexpression, even in the first stages of AD, was demonstrated. Remarkably, FFA3R-/-/APPswe mice do not have the characteristic memory impairment of 12-month-old APPswe mice. Also, this newly generated transgenic line does not develop the most important Alzheimer’s disease (AD)-related features, such as amyloid beta (Aβ) brain accumulations and tau hyperphosphorylation. These findings are accompanied by increased levels of the insulin-degrading enzyme (IDE) and lower activity of the tau kinases GSK3β and Cdk5. We conclude that the brain FFA3R is involved in cognitive processes and its inactivation prevents AD-like cognitive decline and pathological hallmarks.

Genetic Inactivation of D-Amino Acid Oxidase Genes in Methylotrophic Yeast Hansenula Polymorpha

Cellular D-amino acid oxidases (ODA-FAD containing flavoproteins) are widely used in biotechnology. In this regard, it is of particular interest to investigate the functional role of the composition and regulation of individual ODA genes. The aim of this work was to investigate physiological and biochemical characteristics of ODA genes of strain DL1x Hansenula (O.) polymorpha in vivo using gene knockout methodology as well as to determine the nature of these genes expression and regulation of ODA activity as a function of nitrogen and carbon source composition in the cultivation medium. H. polymorpha is a thermotolerant methylotrophic yeast. They are used to study the mechanisms of peroxisomal biogenesis and degradation, regulation of methanol metabolism, nitrate assimilation and stress response. A serial dilution method has been used for rapid assessment of strain growth and stress tolerance. Vector design for genetic inactivation of ODA genes in H. polymorpha was performed using yeast vector pAM773. Selection of "knockout" cell clones was performed using PCR analysis. To complete the transformation process, H. polymorpha was deleted from the pAM773 vector and the obtained DNA was used in the experiment. In vitro cultivation of knockout strains of H. polymorpha was found to exhibit substratespecificity of the ODA. According to the authors, the HP2914 gene is important for D-alanine oxidation, while the gene complex 2400 and 2914 is important for ODA activation in the presence of D-Phe in the medium. It is likely that the gene complex 2082 and 2165 regulates ODA activation when cells are cultured in medium with D-Ala and D-Asp. It was found that in the absence of 2165, 2400, 2914 genes, increased ODA activity to D-Ala only was observed in cell culture medium. The presence of D-alanine combined with 1 % glycerol and 1 % methanol in the culture medium stimulated the activity of the three major ODAs of H. polymorpha through the expression of the HP2914 gene, while the presence of glucose and L-alanine in the culture medium suppressed their activity

The mycobacterial mutasome: composition and recruitment in live cells

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome', minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins, remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

Dissecting Abdominal Aortic Aneurysm Is Aggravated by Genetic Inactivation of LIGHT (TNFSF14)

Abdominal aortic aneurysm (AAA), is a complex disorder characterized by vascular vessel wall remodeling. LIGHT (TNFSF14) is a proinflammatory cytokine associated with vascular disease. In the present study, the impact of genetic inactivation of Light was investigated in dissecting AAA induced by angiotensin II (AngII) in the Apolipoprotein E-deficient (Apoe−/−) mice. Studies in aortic human (ah) vascular smooth muscle cells (VSMC) to study potential translation to human pathology were also performed. AngII-treated Apoe−/−Light−/− mice displayed increased abdominal aorta maximum diameter and AAA severity compared with Apoe−/− mice. Notably, reduced smooth muscle α-actin+ area and Acta2 and Col1a1 gene expression were observed in AAA from Apoe−/−Light−/− mice, suggesting a loss of VSMC contractile phenotype compared with controls. Decreased Opn and augmented Sox9 expression, which are associated with detrimental and non-contractile osteochondrogenic VSMC phenotypes, were also seen in AngII-treated Apoe−/−Light−/− mouse AAA. Consistent with a role of LIGHT preserving VSMC contractile characteristics, LIGHT-treatment of ahVSMCs diminished the expression of SOX9 and of the pluripotency marker CKIT. These effects were partly mediated through lymphotoxin β receptor (LTβR) as the silencing of its gene ablated LIGHT effects on ahVSMCs. These studies suggest a protective role of LIGHT through mechanisms that prevent VSMC trans-differentiation in an LTβR-dependent manner.

Potentiation of amyloid beta phagocytosis and amelioration of synaptic dysfunction upon FAAH deletion in a mouse model of Alzheimer's disease

Background The complex pathophysiology of Alzheimer’s disease (AD) hampers the development of effective treatments. Attempts to prevent neurodegeneration in AD have failed so far, highlighting the need for further clarification of the underlying cellular and molecular mechanisms. Neuroinflammation seems to play a crucial role in disease progression, although its specific contribution to AD pathogenesis remains elusive. We have previously shown that the modulation of the endocannabinoid system (ECS) renders beneficial effects in a context of amyloidosis, which triggers neuroinflammation. In the 5xFAD model, the genetic inactivation of the enzyme that degrades anandamide (AEA), the fatty acid amide hydrolase (FAAH), was associated with a significant amelioration of the memory deficit. Methods In this work, we use electrophysiology, flow cytometry and molecular analysis to evaluate the cellular and molecular mechanisms underlying the improvement associated to the increased endocannabinoid tone in the 5xFAD mouse− model. Results We demonstrate that the chronic enhancement of the endocannabinoid tone rescues hippocampal synaptic plasticity in the 5xFAD mouse model. At the CA3–CA1 synapse, both basal synaptic transmission and long-term potentiation (LTP) of synaptic transmission are normalized upon FAAH genetic inactivation, in a CB1 receptor (CB1R)- and TRPV1 receptor-independent manner. Dendritic spine density in CA1 pyramidal neurons, which is notably decreased in 6-month-old 5xFAD animals, is also restored. Importantly, we reveal that the expression of microglial factors linked to phagocytic activity, such as TREM2 and CTSD, and other factors related to amyloid beta clearance and involved in neuron–glia crosstalk, such as complement component C3 and complement receptor C3AR, are specifically upregulated in 5xFAD/FAAH−/− animals. Conclusion In summary, our findings support the therapeutic potential of modulating, rather than suppressing, neuroinflammation in Alzheimer’s disease. In our model, the long-term enhancement of the endocannabinoid tone triggered augmented microglial activation and amyloid beta phagocytosis, and a consequent reversal in the neuronal phenotype associated to the disease.

Sonic hedgehog is essential for proximal-distal outgrowth of the limb bud in salamanders

The developing forelimb has been a foundational model to understand how specified progenitor cells integrate genetic information to produce the tetrapod limb bauplan (1, 2). Although the reigning hypothesis is that all tetrapods develop limbs in a similar manner, recent work suggests that urodeles have evolved a derived mode of limb development (3-5). Here we demonstrate through pharmacological and genetic inactivation of Sonic hedgehog (Shh) signaling in axolotls that Shh directs expansion and survival of limb progenitor cells in addition to patterning the limb across the proximodistal and antero-posterior axis. In contrast to inactivation of Shh in mouse or chick embryos where a humerus, radius and single digit develop (6-9), Shh crispant axolotls completely lack forelimbs. In rescuing limb development by implanting SHH-N protein beads into the nascent limb field of Shh-crispants, we show that the limb field is specified in the absence of Shh and that hedgehog pathway activation is required to initiate proximodistal outgrowth. When the derived nature of salamander limb development is placed in a phylogenetic context, it generates a new hypothesis where the ability to regenerate an entire tetrapod limb may have evolved uniquely among urodeles.

Redundant mechanisms driven independently by RUNX1 and GATA2 for hematopoietic development

RUNX1 is essential for the generation of hematopoietic stem cells (HSCs). Runx1 null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, even though zebrafish embryos with a runx1 W84X mutation have defects in early definitive hematopoiesis, some runx1W84X/W84X embryos can develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. Here, using three new zebrafish runx1-/- lines we uncovered the compensatory mechanism for runx1-independent hematopoiesis. We show that, in the absence of a functional runx1, a cd41-GFP+ population of hematopoietic precursors still emerge from the hemogenic endothelium and can colonize the hematopoietic tissues of the mutant embryos. Single-cell RNA sequencing of the cd41-GFP+ cells identified a set of runx1-/--specific signature genes during hematopoiesis. Significantly, gata2b, which normally acts upstream of runx1 for the generation of HSCs, was increased in the cd41-GFP+ cells in runx1- /- embryos. Interestingly, genetic inactivation of both gata2b and its paralog, gata2a, did not affect hematopoiesis. However, knocking out runx1 and any three of the four alleles of gata2a and gata2b abolished definitive hematopoiesis. Gata2 expression was also upregulated in hematopoietic cells in Runx1-/- mice, suggesting the compensatory mechanism is conserved. Our findings indicate that RUNX1 and GATA2 serve redundant roles for HSC production, acting as each other's safeguard.

Therapeutic Potential of EWSR1–FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma

Ewing sarcoma is an aggressive bone cancer affecting children and young adults. The main molecular hallmark of Ewing sarcoma are chromosomal translocations that produce chimeric oncogenic transcription factors, the most frequent of which is the aberrant transcription factor EWSR1–FLI1. Because this is the principal oncogenic driver of Ewing sarcoma, its inactivation should be the best therapeutic strategy to block tumor growth. In this study, we genetically inactivated EWSR1–FLI1 using CRISPR-Cas9 technology in order to cause permanent gene inactivation. We found that gene editing at the exon 9 of FLI1 was able to block cell proliferation drastically and induce senescence massively in the well-studied Ewing sarcoma cell line A673. In comparison with an extensively used cellular model of EWSR1–FLI1 knockdown (A673/TR/shEF), genetic inactivation was more effective, particularly in its capability to block cell proliferation. In summary, genetic inactivation of EWSR1–FLI1 in A673 Ewing sarcoma cells blocks cell proliferation and induces a senescence phenotype that could be exploited therapeutically. Although efficient and specific in vivo CRISPR-Cas9 editing still presents many challenges today, our data suggest that complete inactivation of EWSR1–FLI1 at the cell level should be considered a therapeutic approach to develop in the future.