The Combined Effects of Amyloidosis and Serotonin Deficiency by Tryptophan Hydroxylase-2 Knockout Impacts Viability of the APP/PS1 Mouse Model of Alzheimer’s Disease

Background: A decline of brain serotonin (5-HT) is held responsible for the changes in mood that can be observed in Alzheimer’s disease (AD). However, 5-HT’ergic signaling is also suggested to reduce the production of pathogenic amyloid-4β (Aβ). Objective: To investigate the effect of targeted inactivation of tryptophan hydroxylase-2 (Tph2), which is essential for neuronal 5-HT synthesis, on amyloidosis in amyloid precursor protein (APP)swe/presenilin 1 (PS1) ΔE9 transgenic mice. Methods: Triple-transgenic (3xTg) APP/PS1 mice with partial (+/-) or complete Tph2 knockout (–/–) were allowed to survive until 6 months old with APP/PS1, Tph2–/–, and wildtype mice. Survival and weight were recorded. Levels of Aβ 42/40/38, soluble APPα (sAβPPα) and sAβPPβ, and cytokines were analyzed by mesoscale, neurotransmitters by mass spectrometry, and gene expression by quantitative PCR. Tph2, microglia, and Aβ were visualized histologically. Results: Tph2 inactivation in APP/PS1 mice significantly reduced viability, without impacting soluble and insoluble Aβ 42 and Aβ 40 in neocortex and hippocampus, and with only mild changes of soluble Aβ 42/Aβ 40. However, sAβPPα and sAβPPβ in hippocampus and Aβ 38 and Aβ 40 in cerebrospinal fluid were reduced. 3xTg–/–mice were devoid of Tph2 immunopositive fibers and 5-HT. Cytokines were unaffected by genotype, as were neocortical TNF, HTR2a and HTR2b mRNA levels in Tph2–/– mice. Microglia clustered around Aβ plaques regardless of genotype. Conclusion: The results suggest that Tph2 inactivation influences AβPP processing, at least in the hippocampus, although levels of Aβ are unchanged. The reduced viability of 3xTg–/–mice could indicate that 5-HT protects against the seizures that can impact the viability of APP/PS1 mice.

Perturbations in the Heme and Siroheme Biosynthesis Pathways Causing Accumulation of Fluorescent Free Base Porphyrins and Auxotrophy in Ogataea Yeasts

The biosynthesis of cyclic tetrapyrrol chromophores such as heme, siroheme, and chlorophyll involves the formation of fluorescent porphyrin precursors or compounds, which become fluorescent after oxidation. To identify Ogataea polymorpha mutations affecting the final steps of heme or siroheme biosynthesis, we performed a search for clones with fluorescence characteristic of free base porphyrins. One of the obtained mutants was defective in the gene encoding a homologue of Saccharomyces cerevisiae Met8 responsible for the last two steps of siroheme synthesis. Same as the originally obtained mutation, the targeted inactivation of this gene in O. polymorpha and O. parapolymorpha led to increased porphyrin fluorescence and methionine auxotrophy. These features allow the easy isolation of Met8-defective mutants and can potentially be used to construct auxotrophic strains in various yeast species. Besides MET8, this approach also identified the HEM3 gene encoding porphobilinogen deaminase, whose increased dosage led to free base porphyrin accumulation.

Ubiquitination and degradation of NF90 by Tim-3 inhibits antiviral innate immunity

Nuclear Factor 90 (NF90) is a novel virus sensor that serves to initiate antiviral innate immunity by triggering the stress granules (SGs) formation. However, the regulation of the NF90-SGs pathway remain largely unclear. We found that Tim-3, an immune checkpoint inhibitor, promotes the ubiquitination and degradation of NF90 and inhibits NF90-SGs mediated antiviral immunity. Vesicular Stomatitis Virus (VSV) infection induces the up-regulation and activation of Tim-3 in macrophages which in turn recruited the E3 ubiquitin ligase TRIM47 to the zinc finger domain of NF90 and initiated a proteasome-dependent degradation via K48-linked ubiquitination at Lys297. Targeted inactivation of the Tim-3 enhances the NF90 downstream SGs formation by selectively increasing the phosphorylation of PKR and eIF2a, the expression of SGs markers G3BP1 and TIA-1, and protected mice from VSV challenge. These findings provide insights into the crosstalk between Tim-3 and other receptors in antiviral innate immunity and its related clinical significance.

Correction to: Targeted Inactivation of Rin3 Increases Trabecular Bone Mass by Reducing Bone Resorption and Favouring Bone Formation

A correction to this paper has been published: https://doi.org/10.1007/s00223-021-00849-w

Targeted Inactivation of Rin3 Increases Trabecular Bone Mass by Reducing Bone Resorption and Favouring Bone Formation

Common genetic variants at the RIN3 locus on chromosome 14q32 predispose to Paget’s disease of bone (PDB) but the mechanisms by which they do so are unknown. Here, we analysed the skeletal phenotype of female mice with targeted inactivation of the mouse Rin3 gene (Rin3−/−) as compared with wild-type littermates. The Rin3−/− mice had higher trabecular bone volume (BV/TV%) compared with wild type. Mean ± standard deviation values at the distal femur at 8 weeks were 9.0 ± 2.5 vs. 7.0 ± 1.5 (p = 0.002) and at 52 weeks were 15.8 ± 9.5 vs. 8.5 ± 4.2 (p = 0.002). No differences were observed in femoral cortical bone parameters with the exception of marrow diameter which was significantly smaller in 52-week-old Rin3−/− mice compared to wild type: (0.43 mm ± 0.1 vs. 0.57 mm ± 0.2 (p = 0.001). Bone histomorphometry showed a lower osteoclast surface / bone surface (Oc.S/BS%) at 8 weeks in Rin3−/− mice compared to wild type (24.1 ± 4.7 vs. 29.7 ± 6.6; p = 0.025) but there were no significant differences in markers of bone formation at this time. At 52 weeks, Oc.S/BS did not differ between genotypes but single labelled perimeter (SL.Pm/B.Pm (%)) was significantly higher in Rin3−/− mice (24.4 ± 6.4 vs. 16.5 ± 3.8, p = 0.003). We conclude that Rin3 negatively regulates trabecular bone mass in mice by inhibiting osteoclastic bone resorption and favouring bone formation. Our observations also suggest that the variants that predispose to PDB in humans probably do so by causing a gain-in-function of RIN3.

Ubiquitination and degradation of NF90 by Tim-3 inhibits antiviral innate immunity

Nuclear Factor 90 (NF90) is a novel virus sensor that serves to initiate antiviral innate immunity by triggering the stress granules (SGs) formation. However, the regulation of the NF90-SGs pathway remain largely unclear. We found that Tim-3, an immune checkpoint inhibitor, promotes the ubiquitination and degradation of NF90 and inhibits NF90-SGs mediated antiviral immunity. Vesicular Stomatitis Virus (VSV) infection induces the up-regulation and activation of Tim-3 in macrophages which in turn recruited the E3 ubiquitin ligase TRIM47 to the zinc finger domain of NF90 and initiated a proteasome-dependent degradation of the NF90 via K48-linked ubiquitination at Lys297. Targeted inactivation of the Tim-3 enhances the NF90 downstream SGs formation by selectively increasing the phosphorylation of PKR and eIF2a, the expression of SGs markers G3BP1 and TIA-1, and protected mice from lethal VSV challenge. These findings provide insights into the crosstalk between Tim-3 and other receptors in antiviral innate immunity and its related clinical significance.

Designer nucleases to treat malignant cancers driven by viral oncogenes

Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a significant global health burden. Beyond simply causing cellular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that significantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and substantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy. In many of these virus-associated cancers, the prognosis remains extremely poor, and novel drug approaches are urgently needed. Unlike non-specific small-molecule drug screens or the broad-acting toxic effects of chemo- and radiation therapy, the age of designer nucleases permits a rational approach to inactivating disease-causing targets, allowing for permanent inactivation of viral elements to inhibit tumorigenesis with growing evidence to support their efficacy in this role. Although many challenges remain for the clinical application of designer nucleases towards viral oncogenes; the uniqueness and clear molecular mechanism of these targets, combined with the distinct advantages of specific and permanent inactivation by nucleases, argues for their development as next-generation treatments for this aggressive group of cancers.

The Influence of an Elevated Production of Extracellular Enveloped Virions of the Vaccinia Virus on Its Properties in Infected Mice

The modern approach to developing attenuated smallpox vaccines usually consists in targeted inactivation of vaccinia virus (VACV) virulence genes. In this work, we studied how an elevated production of extracellular enveloped virions (EEVs) and the route of mouse infection can influence the virulence and immunogenicity of VACV. The research subject was the LIVP strain, which is used in Russia for smallpox vaccination. Two point mutations causing an elevated production of EEVs compared with the parental LIVP strain were inserted into the sequence of the VACV A34R gene. The created mutant LIVP-A34R strain showed lower neurovirulence in an intracerebral injection test and elevated antibody production in the intradermal injection method. This VACV variant can be a promising platform for developing an attenuated, highly immunogenic vaccine against smallpox and other orthopoxvirus infections. It can also be used as a vector for designing live-attenuated recombinant polyvalent vaccines against various infectious diseases.