The AIM2 inflammasome exacerbates atherosclerosis in clonal haematopoiesis

Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors(1). Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2(V617F) (JAK2(VF)) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease(1,2). Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2(VF) selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2(VF) lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2(VF) lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1 beta reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2(VF) macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1 beta or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk

Transcriptional and Post-Transcriptional Regulation of SPAST, the Gene Most Frequently Mutated in Hereditary Spastic Paraplegia

Hereditary spastic paraplegias (HSPs) comprise a group of neurodegenerative disorders that are characterized by progressive spasticity of the lower extremities, due to axonal degeneration in the corticospinal motor tracts. HSPs are genetically heterogeneous and show autosomal dominant inheritance in ∼70–80% of cases, with additional cases being recessive or X-linked. The most common type of HSP is SPG4 with mutations in the SPAST gene, encoding spastin, which occurs in 40% of dominantly inherited cases and in ∼10% of sporadic cases. Both loss-of-function and dominant-negative mutation mechanisms have been described for SPG4, suggesting that precise or stoichiometric levels of spastin are necessary for biological function. Therefore, we hypothesized that regulatory mechanisms controlling expression of SPAST are important determinants of spastin biology, and if altered, could contribute to the development and progression of the disease. To examine the transcriptional and post-transcriptional regulation of SPAST, we used molecular phylogenetic methods to identify conserved sequences for putative transcription factor binding sites and miRNA targeting motifs in the SPAST promoter and 3′-UTR, respectively. By a variety of molecular methods, we demonstrate that SPAST transcription is positively regulated by NRF1 and SOX11. Furthermore, we show that miR-96 and miR-182 negatively regulate SPAST by effects on mRNA stability and protein level. These transcriptional and miRNA regulatory mechanisms provide new functional targets for mutation screening and therapeutic targeting in HSP

Human Perception Of Noise In Open Spaces In San Juan, Puerto Rico

The Environmental Quality Board (EQB), a government agency in Puerto Rico, is responsible for protecting the environment and quality of life on the island. One area of concern for the EQB is noise pollution. This project analyzed the levels and effects of noise in open spaces, such as parks, in San Juan, Puerto Rico. Surveys were administered to open-space visitors during noise measurements to determine how people perceived various sources and levels of noise at the time of exposure. We investigated whether our measured noise levels comply with current legislation. Finally, we recommended that the EQB improves current regulations and their enforcement, lowers traffic noise, and increases public awareness through education

NRF1 chromatin immunoprecipitation (ChIP) assays.

<p>NRF1 interacts with the <i>SPAST</i> promoter in <b>A)</b> human SK-N-SH cells, and <b>B)</b> murine Neuro2a cells, by ChIP assay. The <i>SNURF</i>-<i>SNRPN</i> enhancer for human and <i>Snurf</i>-<i>Snrpn</i> promoter for mouse are positive controls for <i>cis</i>-elements known to be bound by NRF1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036505#pone.0036505-RodriguezJato1" target="_blank">[89]</a>. Chromatin was immunoprecipitated with anti-NRF1 antibodies, and the promoter regions were assessed by PCR. Controls are no antibody (no Ab) and total input DNA (TI) for ChIP, and a water control (H₂O) for PCR.</p

siRNA targeting <i>NRF1</i> mRNA ablates <i>SPAST</i> promoter function.

<p><b>A</b>) Cartoon showing the structure of the pGL3e-SPAST-promoter-luciferase vector, and the inhibitory mechanisms of siRNA action. Symbols are as for <b>Fig. 1A</b>. <b>B</b>) The plasmid pGL3e-SPAST-promoter was co-transfected into SK-N-SH cells with pSUPER shRNA vectors that target either <i>NRF1</i>, luciferase, or negative control (<i>Arl2</i>). *, <i>P</i><0.05.</p

siRNA targeting <i>NRF1</i> mRNA knocks down <i>SPAST</i>

<p><b>mRNA expression.</b> SK-N-SH cells were transfected with a pSUPER-<i>NRF1</i> shRNA expression vector or mock transfected, with qualitative analysis of gene expression including a negative control (<i>GAPDH</i>). The quantitative band intensities for the “siRNA” and “mock” transfection samples were compared and the ratio is listed to the right of the gel images. The <i>GAPDH</i> mRNA level is unaffected by siRNA targeting <i>NRF1</i>, whereas the mRNA levels for <i>NRF1</i> and <i>SPAST</i> are reduced by the siRNA treatment. The experiment was repeated three times with equivalent results to the representative results shown here.</p

Transcriptional regulation of the <i>SPAST</i> gene encoding spastin (SPG4). A

<p>) Cartoon showing the human <i>SPAST</i> promoter structure with <i>cis</i>-elements representing putative transcription factor (TF) binding sites for NRF1, SOX11, and Sp1. As is typical of CpG-promoters, transcription start sites (TSS) are spread over a large region in exon 1, with two major TSS positions indicated by arrows <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036505#pone.0036505-Mancuso1" target="_blank">[31]</a>. There are two alternative translational initiation codons for spastin 68 and 60 kDa polypeptide isoforms, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036505#pone.0036505-Mancuso1" target="_blank">[31]</a>. <b>B</b>) Multi-sequence alignment of conserved TF <i>cis</i>-elements in representative mammalian species. Sequences were aligned using ClustalW 2.1 and manually adjusted as needed for maximum parsimony. Evolutionarily conserved TF motifs are indicated; *, nucleotide positions conserved in all 19 species; ∧, nucleotide positions conserved in 17/19 species; yellow shading, highly conserved SOX11 motif; red, NRF1 motifs; purple, Sp1 motifs. The NRF1 and SOX11 motifs are highly conserved, but only one Sp1 motif near the 5′ TSS is conserved in mammals. Extended alignments of the complete promoter region into exon 1 and including the first translational start codon are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036505#pone.0036505.s002" target="_blank">Fig. S1</a></b>.</p

Over-expression of SOX11 and NRF1 upregulates endogenous <i>SPAST</i> expression.

<p>Cells were transfected with SOX11 or NRF1 expression vectors and compared to cells transfected with the transfection control (pGL3b). By qRT-PCR, with normalization to <i>GAPDH</i> mRNA levels and to the transfection control, the levels of <i>NRF1</i> and <i>SOX11</i> mRNA were increased 182±28-fold and 2,925±241-fold on transfection with NRF1-VP16 and CMV1-SOX11, respectively (data not shown). Similarly, by qRT-PCR analysis, <i>SPAST</i> transcript levels are significantly increased by over-expression of SOX11 and NRF1. These data represent the average of three biological replicates each done in triplicate. *, <i>P</i><0.05.</p

Regulation of the human <i>SPAST</i> promoter by SOX11.

<p><b>A</b>) Luciferase reporter assays with the human <i>SPAST</i> promoter. The plasmid pGL3b-SPAST-promoter-Luc (third row) and two deletion derivatives (depicted in the panel on the left) were transfected into Flp-in293 cells and normalized to cells transfected with the pGL3b vector (fourth row). *, <i>P</i><0.05. <b>B</b>) Over-expression of SOX11 upregulates <i>SPAST</i> promoter-reporter constructs having a SOX11 binding site. Flp-In-293 cells were transfected with <i>SPAST</i>-promoter-luciferase reporter constructs both with (blue) and without (red) the SOX11 expression vector. *, <i>P</i><0.05; **, <i>P</i><0.001; ***, <i>P</i><0.0001, and n.s., not statistically significant. Symbols in A and B are as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036505#pone-0036505-g001" target="_blank"><b>Fig. 1A</b></a>.</p