Pathways Regulating Inflammation in Microglia and Ageing

PhDInflammation is implicated in a wide array of diseases and is associated with the ageing process: ‘inflammageing’ is the low-grade inflammation that occurs as an organism ages. I was particularly interested in age-related inflammation of the brain, thought to be mediated by microglia, the immune cells of the central nervous system (CNS). To this end, I carried out RNA sequencing of microglia from young (6 months) and aged mice (23 months). I found that microglia from aged mice have a very distinct transcriptome signature. Interestingly, pathways associated with mTOR signalling and inflammation were upregulated. Given the evidence that mTOR is a key modulator of ageing, I investigated its role in inflammation in microglia. Using a mouse model in which Rheb, a positive regulator of mTORC1, was knocked out in Csf1r-expressing cells (microglia and macrophages), I found that in vivo LPS stimulation caused a significant increase in the transcription of inflammatory genes in microglia from mTORC1-deficient mice compared to controls. The effect was further exaggerated in mTORC1-deficient aged mice, suggesting a role for mTORC1 in the priming of aged microglia. However, these transcriptome changes were not translated into protein; indeed, Csf1r-Cre; Rheb f/f mice showed reduced overall inflammation, as measured by sickness behaviour in the openfield test and by plasma cytokine levels. On the other hand, long-term treatment with rapamycin in vivo showed a very distinct phenotype, with reduced inflammation following LPS stimulation in young mice but no effect in aged ones. This PhD thesis sheds new light on pathways regulating microglia in ageing and has clinical implications for pathologies in which inflammation plays a major role.Age Uk

Predicting optical coherence tomography-derived diabetic macular edema grades from fundus photographs using deep learning

Diabetic eye disease is one of the fastest growing causes of preventable blindness. With the advent of anti-VEGF (vascular endothelial growth factor) therapies, it has become increasingly important to detect center-involved diabetic macular edema (ci-DME). However, center-involved diabetic macular edema is diagnosed using optical coherence tomography (OCT), which is not generally available at screening sites because of cost and workflow constraints. Instead, screening programs rely on the detection of hard exudates in color fundus photographs as a proxy for DME, often resulting in high false positive or false negative calls. To improve the accuracy of DME screening, we trained a deep learning model to use color fundus photographs to predict ci-DME. Our model had an ROC-AUC of 0.89 (95% CI: 0.87-0.91), which corresponds to a sensitivity of 85% at a specificity of 80%. In comparison, three retinal specialists had similar sensitivities (82-85%), but only half the specificity (45-50%, p<0.001 for each comparison with model). The positive predictive value (PPV) of the model was 61% (95% CI: 56-66%), approximately double the 36-38% by the retinal specialists. In addition to predicting ci-DME, our model was able to detect the presence of intraretinal fluid with an AUC of 0.81 (95% CI: 0.81-0.86) and subretinal fluid with an AUC of 0.88 (95% CI: 0.85-0.91). The ability of deep learning algorithms to make clinically relevant predictions that generally require sophisticated 3D-imaging equipment from simple 2D images has broad relevance to many other applications in medical imaging

SETD2 transcriptional control of ATG14L/S isoforms regulates autophagosome-lysosome fusion

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms.Peer reviewe

mTOR-dependent translation amplifies microglia priming in aging mice.

peer reviewedMicroglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB-dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Assessing mRNA translation in mouse adult microglia and bone-marrow-derived macrophages

Summary: Protein synthesis, or mRNA translation, is the biological process through which genetic information stored in messenger RNAs is encoded into proteins. Here, we present an optimized protocol for assessing the translation rate in mouse adult microglia and cultured bone-marrow-derived macrophages. We describe steps for isolating cells, treating them with a puromycin-analog probe, and fluorescently labeling the puromycylated-polypeptide chains. We then detail their quantification by flow cytometry or with a fluorescent plate reader.For complete details on the use and execution of this protocol, please refer to Keane et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

A two-stage social network intervention for reducing alcohol and other drug use in residential colleges : Protocol for a feasibility trial

Background: In many countries, the rate of binge drinking and alcohol-related harms among students living in residential colleges exceeds that observed among young people in the general population. Peer influence plays a key role in driving alcohol and other drug (AOD) use in colleges. This highlights the potential role of peer influence AOD-interventions in college student-networks. This protocol paper outlines the design of a two-stage social network intervention (SNI) for reducing AOD-use in four Australian first-year residential college networks. Methods: In Stage 1, a peer-led workshop will provide education about AOD-use and harm-minimisation strategies to four first-year cohorts in the first week of semester one (N ~ 500). In Stage 2, a targeted SNI will be delivered to the largest co-educational, first-year cohort (N ~ 160), wherein heavy drinking 'Strategic Players' (influential students) will be identified and offered a brief, telephone-delivered motivational interviewing intervention for AOD-use (QuikFix). Participants will complete online surveys at baseline and 12-, 26-, and 52- weeks follow-up. Results: Recruitment occurred in February 2021 and is now closed. Results are expected to be submitted for publication in late 2022. Conclusions: This protocol paper outlines the design of a feasibility trial exploring the impact of applied SNIs for reducing AOD-use and related consequences in residential college student networks. If effective, the two-stage SNI proposed could (i) reduce AOD-use and risk of harm across first-year student networks and (ii) provide an effective brief intervention (QuikFix) to high-risk drinkers who have greatest potential to spread the intervention effects to other risky drinkers in their network. Trial registration: Australian New Zealand Clinical Trials Registry (ACTRN12621000494831)

Cell Death Dis

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms