Membrane-type 5-matrix metalloproteinase (MT5-MMP) is a proteinase mainly expressed in the nervous system with emerging roles in brain pathophysiology. The implication of MT5-MMP in Alzheimer’s disease (AD), notably its interplay with the amyloidogenic process, remains elusive. Accordingly, we crossed the genetically engineered 5xFAD mouse model of AD with MT5-MMP-deficient mice and examined the impact of MT5-MMP deficiency in bigenic 5xFAD/MT5-MMP−/− mice. At early stages (4 months) of the pathology, the levels of amyloid beta peptide (Aβ) and its amyloid precursor protein (APP) C-terminal fragment C99 were largely reduced in the cortex and hippocampus of 5xFAD/MT5-MMP−/−, compared to 5xFAD mice. Reduced amyloidosis in bigenic mice was concomitant with decreased glial reactivity and interleukin-1β (IL-1β) levels, and the preservation of long-term potentiation (LTP) and spatial learning, without changes in the activity of α-, β- and γ-secretases. The positive impact of MT5-MMP deficiency was still noticeable at 16 months of age, as illustrated by reduced amyloid burden and gliosis, and a better preservation of the cortical neuronal network and synaptophysin levels in bigenic mice. MT5-MMP expressed in HEKswe cells colocalized and co-immunoprecipitated with APP and significantly increased the levels of Aβ and C99. MT5-MMP also promoted the release of a soluble APP fragment of 95 kDa (sAPP95) in HEKswe cells. sAPP95 levels were significantly reduced in brain homogenates of 5xFAD/MT5-MMP−/− mice, supporting altogether the idea that MT5-MMP influences APP processing. MT5-MMP emerges as a new pro-amyloidogenic regulator of APP metabolism, whose deficiency alleviates amyloid pathology, neuroinflammation and cognitive decline.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-015-1992-1) contains supplementary material, which is available to authorized users.

Transcription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the conservation of introns throughout the eukaryotic lineage.

Matrix metalloproteinases (MMPs) are pleiotropic endopeptidases involved in a variety of neurodegenerative/neuroinflammatory processes through their interactions with a large number of substrates. Among those, the amyloid precursor protein (APP) and the beta amyloid peptide (Aβ) are largely associated with the development of Alzheimer’s disease (AD). However, the regulation and potential contribution of MMPs to AD remains unclear. In this study, we investigated the evolution of the expression of MMP-2, MMP-9, and membrane-type 1-MMP (MT1-MMP) in the hippocampus at different stages of the pathology (asymptomatic, prodromal-like and symptomatic) in the 5xFAD transgenic mouse AD model. In parallel we also followed the expression of functionally associated factors. Overall, the expression of MMP-2, MMP-9, and MT1-MMP was upregulated concomitantly with the tissue inhibitor of MMPs-1 (TIMP-1) and several markers of inflammatory/glial response. The three MMPs exhibited age- and cell-dependent upregulation of their expression, with MMP-2 and MMP-9 being primarily located to astrocytes, and MT1-MMP to neurons. MMP-9 and MT1-MMP were also prominently present in amyloid plaques. The levels of active MT1-MMP were highly upregulated in membrane-enriched fractions of hippocampus at 6 months of age (symptomatic phase), when the levels of APP, its metabolites APP C-terminal fragments (CTFs), and Aβ trimers were the highest. Overexpression of MT1-MMP in HEK cells carrying the human APP Swedish mutation (HEKswe) strongly increased β-secretase derived C-terminal APP fragment (C99) and Aβ levels, whereas MMP-2 overexpression nearly abolished Aβ production without affecting C99. Our data consolidate the emerging idea of a regulatory interplay between MMPs and the APP/Aβ system, and demonstrate for the first time the pro-amyloidogenic features of MT1-MMP. Further investigation will be justified to evaluate this MMP as a novel potential therapeutic target in AD.

Transposable elements are in a constant arms race with the silencing mechanisms of their host genomes. One silencing mechanism commonly used by many eukaryotes is dependent on cytosine methylation, a covalent modification of DNA deposited by C5 cytosine methyltransferases (DNMTs). Here, we report how two distantly related eukaryotic lineages, dinoflagellates and charophytes, have independently incorporated DNMTs into the coding regions of distinct retrotransposon classes. Concomitantly, we show that dinoflagellates of the genus Symbiodinium have evolved cytosine methylation patterns unlike any other eukaryote, with most of the genome methylated at CG dinucleotides. Finally, we demonstrate the ability of retrotransposon DNMTs to methylate CGs de novo, suggesting that retrotransposons could self-methylate retrotranscribed DNA. Together, this is an example of how retrotransposons incorporate host-derived genes involved in DNA methylation. In some cases, this event could have implications for the composition and regulation of the host epigenomic environment.

We previously reported that deficiency of membrane-type five matrix metalloproteinase (MT5-MMP) prevents amyloid pathology in the cortex and hippocampus of 5xFAD mice, and ameliorates the functional outcome. We have now investigated whether the integrity of another important area affected in Alzheimer’s disease (AD), the frontal cortex, was also preserved upon MT5-MMP deficiency in 4-month old mice at prodromal stages of the pathology. We used the olfactory H-maze (OHM) to show that learning impairment associated with dysfunctions of the frontal cortex in 5xFAD was prevented in bigenic 5xFAD/MT5-MMP−/− mice. The latter exhibited concomitant drastic reductions of amyloid beta peptide (Aβ) assemblies (soluble, oligomeric and fibrillary) and its immediate precursor, C99. Simultaneously, astrocyte reactivity and tumor necrosis factor alpha (TNF-α) levels were also lowered. Moreover, MT5-MMP deficiency induced a decrease in N-terminal soluble fragments of amyloid precursor protein (APP), including soluble APPα (sAPPα), sAPPβ and the MT5-MMP-linked fragment of 95 kDa, sAPP95. However, the lack of MT5-MMP did not affect the activity of β- and γ-secretases. In cultured HEKswe cells, transiently expressed MT5-MMP localized to early endosomes and increased the content of APP and Aβ40 in these organelles, as well as Aβ levels in cell supernatants. This is the first evidence that the pro-amyloidogenic features of MT5-MMP lie, at least in part, on the ability of the proteinase to promote trafficking into one of the amyloidogenic subcellular loci. Together, our data further support the pathogenic role of MT5-MMP in AD and that its inhibition improves the functional and pathological outcomes, in this case in the frontal cortex. These data also support the idea that MT5-MMP could become a novel therapeutic target in AD.

Integration of transposable elements into the genome is mutagenic. Mechanisms targeting integrations into relatively safe locations, hence minimizing deleterious consequences for cell fitness, have emerged during evolution. In budding yeast, integration of the Ty1 LTR retrotransposon upstream of RNA polymerase III (Pol III)‐transcribed genes requires interaction between Ty1 integrase (IN1) and AC40, a subunit common to Pol I and Pol III. Here, we identify the Ty1 targeting domain of IN1 that ensures (i) IN1 binding to Pol I and Pol III through AC40, (ii) IN1 genome‐wide recruitment to Pol I‐ and Pol III‐transcribed genes, and (iii) Ty1 integration only at Pol III‐transcribed genes, while IN1 recruitment by AC40 is insufficient to target Ty1 integration into Pol I‐transcribed genes. Swapping the targeting domains between Ty5 and Ty1 integrases causes Ty5 integration at Pol III‐transcribed genes, indicating that the targeting domain of IN1 alone confers Ty1 integration site specificity.

Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest—in this case CB2—but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.

Several nuclear pore-associated factors, including the SUMO-protease Ulp1, have been proposed to prevent the export of intron-containing messenger ribonucleoparticles (mRNPs) in yeast. However, the molecular mechanisms of this nuclear pore-dependent mRNA quality control, including the sumoylated targets of Ulp1, have remained unidentified. Here, we demonstrate that the apparent ‘pre-mRNA leakage’ phenotype arising upon ULP1 inactivation is shared by sumoylation mutants of the THO complex, an early mRNP biogenesis factor. Importantly, we establish that alteration of THO complex activity differentially impairs the expression of intronless and intron-containing reporter genes, rather than triggering bona fide ‘pre-mRNA leakage’. Indeed, we show that the presence of introns within THO target genes attenuates the effect of THO inactivation on their transcription. Epistasis analyses further clarify that different nuclear pore components influence intron-containing gene expression at distinct stages. Ulp1, whose maintenance at nuclear pores depends on the Nup84 complex, impacts on THO-dependent gene expression, whereas the nuclear basket-associated Mlp1/Pml39 proteins prevent pre-mRNA export at a later stage, contributing to mRNA quality control. Our study thus highlights the multiplicity of mechanisms by which nuclear pores contribute to gene expression, and further provides the first evidence that intronic sequences can alleviate early mRNP biogenesis defects.