cGAS-STING drives ageing-related inflammation and neurodegeneration.

Low-grade inflammation is a hallmark of old age and a central driver of ageing-associated impairment and disease1. Multiple factors can contribute to ageing-associated inflammation2; however, the molecular pathways that transduce aberrant inflammatory signalling and their impact in natural ageing remain unclear. Here we show that the cGAS-STING signalling pathway, which mediates immune sensing of DNA3, is a critical driver of chronic inflammation and functional decline during ageing. Blockade of STING suppresses the inflammatory phenotypes of senescent human cells and tissues, attenuates ageing-related inflammation in multiple peripheral organs and the brain in mice, and leads to an improvement in tissue function. Focusing on the ageing brain, we reveal that activation of STING triggers reactive microglial transcriptional states, neurodegeneration and cognitive decline. Cytosolic DNA released from perturbed mitochondria elicits cGAS activity in old microglia, defining a mechanism by which cGAS-STING signalling is engaged in the ageing brain. Single-nucleus RNA-sequencing analysis of microglia and hippocampi of a cGAS gain-of-function mouse model demonstrates that engagement of cGAS in microglia is sufficient to direct ageing-associated transcriptional microglial states leading to bystander cell inflammation, neurotoxicity and impaired memory capacity. Our findings establish the cGAS-STING pathway as a driver of ageing-related inflammation in peripheral organs and the brain, and reveal blockade of cGAS-STING signalling as a potential strategy to halt neurodegenerative processes during old age.

8-hydroxy-2'-deoxyguanosine and cardiovascular disease: a systematic review.

Oxidative stress due to an excess of reactive oxygen species (ROS) may play a role in the development and progression of cardiovascular disease (CVD). 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a marker of oxidative DNA damage caused by ROS. This review aimed to assess the association between 8-OHdG and CVD by reviewing the literature. Studies in human subjects using either plasma or urine to determine 8-OHdG concentrations were surveyed. Eighteen relevant studies were found, of which 13 were case-control studies and five had a prospective design. Without exception, the case-control studies showed significant positive associations between 8-OHdG and CVD. In agreement, two prospective studies showed a significant association of 8-OHdG and heart failure. Furthermore, two prospective studies found a significant association between 8-OHdG and stroke, and finally, one prospective study showed a borderline significant (p = 0.08) association between coronary artery disease (CAD) patients developing a cardiac event and 8-OHdG concentrations. In conclusion, high levels of 8-OHdG in blood and urine are associated with atherosclerosis and heart failure, but further large prospective studies are needed to investigate 8-OHdG as a predictor for cardiovascular diseases.

A Lamb1Dendra2 mouse model identifies basement-membrane-producing origins and dynamics in PyMT breast tumors.

The basement membrane (BM) around tumor lobes forms a barrier to prevent cancer cells from invading the surrounding tissue. Although myoepithelial cells are key producers of the healthy mammary epithelium BM, they are nearly absent in mammary tumors. To study the origin and dynamics of the BM, we developed and imaged a laminin beta1-Dendra2 mouse model. We show that the turnover of laminin beta1 is faster in the BMs that surround the tumor lobes than in the BMs that surround the healthy epithelium. Moreover, we find that epithelial cancer cells and tumor-infiltrating endothelial cells synthesize laminin beta1 and that this production is temporarily and locally heterogeneous, leading to local discontinuity of the BM laminin beta1. Collectively, our data draw a new paradigm for tumor BM turnover in which the disassembly happens at a constant rate, and a local misbalance of compensating production leads to reduction or even complete disappearance of the BM.

FIRRM/C1orf112 mediates resolution of homologous recombination intermediates in response to DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) pose a major obstacle for DNA replication and transcription if left unrepaired. The cellular response to ICLs requires the coordination of various DNA repair mechanisms. Homologous recombination (HR) intermediates generated in response to ICLs, require efficient and timely conversion by structure-selective endonucleases. Our knowledge on the precise coordination of this process remains incomplete. Here, we designed complementary genetic screens to map the machinery involved in the response to ICLs and identified FIRRM/C1orf112 as an indispensable factor in maintaining genome stability. FIRRM deficiency leads to hypersensitivity to ICL-inducing compounds, accumulation of DNA damage during S-G2 phase of the cell cycle, and chromosomal aberrations, and elicits a unique mutational signature previously observed in HR-deficient tumors. In addition, FIRRM is recruited to ICLs, controls MUS81 chromatin loading, and thereby affects resolution of HR intermediates. FIRRM deficiency in mice causes early embryonic lethality and accelerates tumor formation. Thus, FIRRM plays a critical role in the response to ICLs encountered during DNA replication.

Inhibition of carnitine palmitoyl-transferase 1 is a potential target in a mouse model of Parkinson's disease.

Glucose metabolism is dysregulated in Parkinson's disease (PD) causing a shift toward the metabolism of lipids. Carnitine palmitoyl-transferase 1A (CPT1A) regulates the key step in the metabolism of long-chain fatty acids. The aim of this study is to evaluate the effect of downregulating CPT1, either genetically with a Cpt1a P479L mutation or medicinally on PD using chronic rotenone mouse models using C57Bl/6J and Park2 knockout mice. We show that Cpt1a P479L mutant mice are resistant to rotenone-induced PD, and that inhibition of CPT1 is capable of restoring neurological function, normal glucose metabolism, and alleviate markers of PD in the midbrain. Furthermore, we show that downregulation of lipid metabolism via CPT1 alleviates pathological motor and non-motor behavior, oxidative stress, and disrupted glucose homeostasis in Park2 knockout mice. Finally, we confirm that rotenone induces gut dysbiosis in C57Bl/6J and, for the first time, in Park2 knockout mice. We show that this dysbiosis is alleviated by the downregulation of the lipid metabolism via CPT1.

QPCTL regulates macrophage and monocyte abundance and inflammatory signatures in the tumor microenvironment.

The enzyme glutaminyl-peptide cyclotransferase-like protein (QPCTL) catalyzes the formation of pyroglutamate residues at the NH2-terminus of proteins, thereby influencing their biological properties. A number of studies have implicated QPCTL in the regulation of chemokine stability. Furthermore, QPCTL activity has recently been shown to be critical for the formation of the high-affinity SIRPα binding site of the CD47 "don't-eat-me" protein. Based on the latter data, interference with QPCTL activity -and hence CD47 maturation-may be proposed as a means to promote anti-tumor immunity. However, the pleiotropic activity of QPCTL makes it difficult to predict the effects of QPCTL inhibition on the tumor microenvironment (TME). Using a syngeneic mouse melanoma model, we demonstrate that QPCTL deficiency alters the intra-tumoral monocyte-to-macrophage ratio, results in a profound increase in the presence of pro-inflammatory cancer-associated fibroblasts (CAFs) relative to immunosuppressive TGF-ß1-driven CAFs, and leads to an increased IFN and decreased TGF-ß transcriptional response signature in tumor cells. Importantly, the functional relevance of the observed TME remodeling is demonstrated by the synergy between QPCTL deletion and anti PD-L1 therapy, sensitizing an otherwise refractory melanoma model to anti-checkpoint therapy. Collectively, these data provide support for the development of strategies to interfere with QPCTL activity as a means to promote tumor-specific immunity.

Actin maturation requires the ACTMAP/C19orf54 protease.

Protein synthesis generally starts with a methionine that is removed during translation. However, cytoplasmic actin defies this rule because its synthesis involves noncanonical excision of the acetylated methionine by an unidentified enzyme after translation. Here, we identified C19orf54, named ACTMAP (actin maturation protease), as this enzyme. Its ablation resulted in viable mice in which the cytoskeleton was composed of immature actin molecules across all tissues. However, in skeletal muscle, the lengths of sarcomeric actin filaments were shorter, muscle function was decreased, and centralized nuclei, a common hallmark of myopathies, progressively accumulated. Thus, ACTMAP encodes the missing factor required for the synthesis of mature actin and regulates specific actin-dependent traits in vivo.

Downregulating carnitine palmitoyl transferase 1 affects disease progression in the SOD1 G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease characterized by death of motor neurons. The etiology and pathogenesis remains elusive despite decades of intensive research. Herein, we report that dysregulated metabolism plays a central role in the SOD1 G93A mouse model mimicking ALS. Specifically, we report that the activity of carnitine palmitoyl transferase 1 (CPT1) lipid metabolism is associated with disease progression. Downregulation of CPT1 activity by pharmacological and genetic methods results in amelioration of disease symptoms, inflammation, oxidative stress and mitochondrial function, whereas upregulation by high-fat diet or corticosterone results in a more aggressive disease progression. Finally, we show that downregulating CPT1 shifts the gut microbiota communities towards a protective phenotype in SOD1 G93A mice. These findings reveal that metabolism, and specifically CPT1 lipid metabolism plays a central role in the SOD1 G93A mouse model and shows that CPT1 might be a therapeutic target in ALS.

Dysregulation of metabolic pathways by carnitine palmitoyl-transferase 1 plays a key role in central nervous system disorders: experimental evidence based on animal models.

The etiology of CNS diseases including multiple sclerosis, Parkinson's disease and amyotrophic lateral sclerosis remains elusive despite decades of research resulting in treatments with only symptomatic effects. In this study, we provide evidence that a metabolic shift from glucose to lipid is a key mechanism in neurodegeneration. We show that, by downregulating the metabolism of lipids through the key molecule carnitine palmitoyl transferase 1 (CPT1), it is possible to reverse or slowdown disease progression in experimental models of autoimmune encephalomyelitis-, SOD1G93A and rotenone models, mimicking these CNS diseases in humans. The effect was seen both when applying a CPT1 blocker or by using a Cpt1a P479L mutant mouse strain. Furthermore, we show that diet, epigenetics, and microbiota are key elements in this metabolic shift. Finally, we present a systemic model for understanding the complex etiology of neurodegeneration and how different regulatory systems are interconnected through a central metabolic pathway that becomes deregulated under specific conditions.

Haploid genetic screens identify SPRING/C12ORF49 as a determinant of SREBP signaling and cholesterol metabolism.

The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBP Regulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRINGKO cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRINGKO cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling.

CPT1A plays a key role in the development and treatment of multiple sclerosis and experimental autoimmune encephalomyelitis.

Human mutations in carnitine palmitoyl transferase 1A (CPT1A) are correlated with a remarkably low prevalence of multiple sclerosis (MS) in Inuits (P479L) and Hutterites (G710E). To elucidate the role of CPT1A, we established a Cpt1a P479L mouse strain and evaluated its sensitivity to experimental autoimmune encephalomyelitis (EAE) induction. Since CPT1a is a key molecule in lipid metabolism, we compared the effects of a high-fat diet (HFD) and normal diet (ND) on disease progression. The disease severity increased significantly in WT mice compared to that in Cpt1 P479L mice. In addition, WT mice receiving HFD showed markedly exacerbated disease course when compared either with Cpt1a P479L mice receiving HFD or WT control group receiving ND. Induction of EAE caused a significant decrease of myelin basic protein expression in the hindbrain of disease affected WT mice in comparison to Cpt1a P479L mice. Further, WT mice showed increased expression of oxidative stress markers like Nox2 and Ho-1, whereas expression of mitochondrial antioxidants regulator Pgc1α was increased in Cpt1a P479L mice. Our results suggest that, lipids metabolism play an important role in EAE, as shown by the higher severity of disease progression in both WT EAE and WT EAF HFD-fed mice in contrast to their counterpart Cpt1a P479L mutant mice. Interestingly, mice with downregulated lipid metabolism due to the Cpt1a P479L mutation showed resistance to EAE induction. These findings support a key role for CPT1A in the development of EAE and could be a promising target in MS treatment.

Direct Generation of Conditional Alleles Using CRISPR/Cas9 in Mouse Zygotes.

Conditional alleles in genetically modified mice allow for the deletion of a gene of interest in a target tissue when combined with a tissue-specific Cre recombinase. A conditional allele is achieved by introducing LoxP sites around a critical exon, a gene, or a cluster of genes. Previously, conditional alleles were introduced in the mouse germline by classic gene targeting in embryonic stem cells, a challenging and time-consuming procedure. Now, conditional alleles can be generated directly in fertilized mouse eggs (zygotes) using the CRISPR/Cas9 technology. This one-step generation of mice is easier in design and faster. Here, we describe our achieved success rate, the considerations in design of a conditional allele, a detailed protocol to prepare the zygote injection mix, and the screening procedure to identify the new conditional knockout mouse strain.