Pathophysiology of cerebral ischaemia: effects of APOE genotype on outcome and endocytosis

Apolipoprotein E (apoE denotes protein: APOE denotes gene) is a lipid-transport protein abundantly expressed in the brain and strongly upregulated after acute brain injury. The APOE e4 allele is the major genetic risk factor for Alzheimer’s disease (AD) and has been associated with poorer outcome after various types of acute brain injury, including traumatic brain injury and subarachnoid haemorrhage. However, the role of APOE genotype in focal ischaemic stroke is less clear. The mechanism(s) by which APOE genotype may modulate outcome after acute brain injury are also unclear at present. Accordingly, the studies described in this thesis were undertaken to further address these issues. 1. Endocytic pathway alterations in human temporal lobe after global cerebral ischaemia and association with APOE genotype. 2. Characterisation and validation of the intraluminal filament model of focal cerebral ischaemia in C57BI/6J mice. 3. Association between APOE genotype and differences in outcome and endocytic pathway alterations after focal cerebral ischaemia in mice. 4. Adenovirus-mediated gene transfer of APOE e3 markedly reduces ischaemic brain damage after focal cerebral ischaemia in mice. The data presented in this thesis indicate an important role for APOE genotype in modulating outcome after ischaemic brain injury, further highlighting the favourable effects associated with the APOE e3 allele. APOE genotype-dependent alterations in the endocytic pathway are mechanisms which could contribute to differences in outcome. These data also highlight the neuroprotective effects achieved by manipulating apoE levels to promote the beneficial effects of apoE3. An apoE-based therapeutic strategy may be a potential approach for treatment of ischaemic brain injury in humans

Nuclear retention of IL-1 alpha by necrotic cells: A mechanism to dampen sterile inflammation

Sterile inflammation is a host response to tissue injury that is mediated by damage-associated molecular patterns (DAMPs) released from dead cells. Sterile inflammation worsens damage in a number of injury paradigms. The pro-inflammatory cytokine interleukin-1α (IL-1α) is reported to be a DAMP released from dead cells, and is known to exacerbate brain injury caused by stroke. In the brain, IL-1α is produced by microglia, the resident brain macrophages. We found that IL-1α is actively trafficked to the nuclei of microglia, and hence tested the hypothesis that trafficking of IL-1α to the nucleus would inhibit its release following necrotic cell death, limiting sterile inflammation. Microglia subjected to oxygen-glucose deprivation (OGD) died via necrosis. Under these conditions, microglia expressing nuclear IL-1α released significantly less IL-1α than microglia with predominantly cytosolic IL-1α. The remaining IL-1α was immobilised in the nuclei of the dead cells. Thus, nuclear retention of IL-1α may serve to limit inflammation following cell death

Activation of Nrf2 to optimise immune responses to intracerebral haemorrhage

Haemorrhage into the brain parenchyma can be devastating. This manifests as spontaneous intracerebral haemorrhage (ICH) after head trauma, and in the context of vascular dementia. Randomised controlled trials have not reliably shown that haemostatic treatments aimed at limiting ICH haematoma expansion and surgical approaches to reducing haematoma volume are effective. Consequently, treatments to modulate the pathophysiological responses to ICH, which may cause secondary brain injury, are appealing. Following ICH, microglia and monocyte derived cells are recruited to the peri-haematomal environment where they phagocytose haematoma breakdown products and secrete inflammatory cytokines, which may trigger both protective and harmful responses. The transcription factor Nrf2, is activated by oxidative stress, is highly expressed by central nervous system microglia and macroglia. When active, Nrf2 induces a transcriptional programme characterised by increased expression of antioxidant, haem and heavy metal detoxification and proteostasis genes, as well as suppression of proinflammatory factors. Therefore, Nrf2 activation may facilitate adaptive-protective immune cell responses to ICH by boosting resistance to oxidative stress and heavy metal toxicity, whilst limiting harmful inflammatory signalling, which can contribute to further blood brain barrier dysfunction and cerebral oedema. In this review, we consider the responses of immune cells to ICH and how these might be modulated by Nrf2 activation. Finally, we propose potential therapeutic strategies to harness Nrf2 to improve the outcomes of patients with ICH

Myeloid Cell and Transcriptome Signatures Associated With Inflammation Resolution in a Model of Self-Limiting Acute Brain Inflammation

<p>Inflammation contributes to tissue repair and restoration of function after infection or injury. However, some forms of inflammation can cause tissue damage and disease, particularly if inappropriately activated, excessive, or not resolved adequately. The mechanisms that prevent excessive or chronic inflammation are therefore important to understand. This is particularly important in the central nervous system where some effects of inflammation can have particularly harmful consequences, including irreversible damage. An increasing number of neurological disorders, both acute and chronic, and their complications are associated with aberrant neuroinflammatory activity. Here we describe a model of self-limiting acute brain inflammation optimized to study mechanisms underlying inflammation resolution. Inflammation was induced by intracerebral injection of lipopolysaccharide (LPS) and the temporal profile of key cellular and molecular changes were defined during the progression of the inflammatory response. The kinetics of accumulation and loss of neutrophils in the brain enabled well-demarcated phases of inflammation to be operatively defined, including induction and resolution phases. Microglial reactivity and accumulation of monocyte-derived macrophages were maximal at the onset of and during the resolution phase. We profiled the transcriptome-wide gene expression changes at representative induction and resolution timepoints and used gene coexpression network analysis to identify gene clusters. This revealed a distinct cluster of genes associated with inflammation resolution that were induced selectively or maximally during this phase and indicated an active programming of gene expression that may drive resolution as has been described in other tissues. Induction of gene networks involved in lysosomal function, lipid metabolism, and a comparative switch to MHC-II antigen presentation (relative to MHC-I during induction) were prominent during the resolution phase. The restoration and/or further induction of microglial homeostatic signature genes was notable during the resolution phase. We propose the current model as a tractable reductionist system to complement more complex models for further understanding how inflammation resolution in the brain is regulated and as a platform for in vivo testing/screening of candidate resolution-modifying interventions. Our data highlight how resolution involves active cellular and transcriptome reprogramming and identify candidate gene networks associated with resolution-phase adaptations that warrant further study.</p

Divergent neuroinflammatory regulation of microglial TREM expression and involvement of NF-κB

The triggering receptor expressed on myeloid cells (TREM) family of proteins are cell surface receptors with important roles in regulation of myeloid cell inflammatory activity. In the central nervous system, TREM2 is implicated in further roles in microglial homeostasis, neuroinflammation and neurodegeneration. Different TREM receptors appear to have contrasting roles in controlling myeloid immune activity therefore the relative and co-ordinated regulation of their expression is important to understand but is currently poorly understood. We sought to determine how microglial TREM expression is affected under neuroinflammatory conditions in vitro and in vivo. Our data show that microglial Trem1 and Trem2 gene expression are regulated in an opposing manner by lipopolysaccharide (LPS) in vitro in both adult murine and human microglia. LPS caused a significant induction of Trem1 and a contrasting suppression of Trem2 expression. We also observed similar divergent Trem1 and Trem2 responses in vivo in response to acute brain inflammation and acute cerebral ischaemia. Our data show that inhibition of NF-κB activation prevents the LPS-induced alterations in both Trem1 and Trem2 expression in vitro indicating NF-κB as a common signaling intermediate controlling these divergent responses. Distinct patterns of microglial Trem1 induction and Trem2 suppression to different Toll-like receptor (TLR) ligands were also evident, notably with Trem1 induction restricted to those ligands activating TLRs signaling via TRIF. Our data show co-ordinated but divergent regulation of microglial TREM receptor expression with a central role for NF-κB. Neuroinflammatory conditions that alter the balance in TREM expression could therefore be an important influence on microglial inflammatory and homeostatic activity with implications for neuroinflammatory and neurodegenerative disease

Defining the pig microglial transcriptome reveals its core signature, regional heterogeneity, and similarity with human and rodent microglia:Pig microglial transcriptome signature

Microglia play key roles in brain homeostasis as well as responses to neurodegeneration and neuroinflammatory processes caused by physical disease and psychosocial stress. The pig is a physiologically relevant model species for studying human neurological disorders, many of which are associated with microglial dysfunction. Furthermore, pigs are an important agricultural species, and there is a need to understand how microglial function affects their welfare. As a basis for improved understanding to enhance biomedical and agricultural research, we sought to characterize pig microglial identity at genome-wide scale and conduct inter-species comparisons. We isolated pig hippocampal tissue and microglia from frontal cortex, hippocampus, and cerebellum, as well as alveolar macrophages from the lungs and conducted RNA-sequencing (RNAseq). By comparing the transcriptomic profiles between microglia, macrophages, and hippocampal tissue, we derived a set of 239 highly enriched genes defining the porcine core microglial signature. We found brain regional heterogeneity based on 150 genes showing significant (adjusted p < 0.01) regional variations and that cerebellar microglia were most distinct. We compared normalized gene expression for microglia from human, mice and pigs using microglia signature gene lists derived from each species and demonstrated that a core microglial marker gene signature is conserved across species, but that species-specific expression subsets also exist. Our data provide a valuable resource defining the pig microglial transcriptome signature that validates and highlights pigs as a useful large animal species bridging between rodents and humans in which to study the role of microglia during homeostasis and disease