“Integrative genomics study of microglial transcriptome reveals effect of DLG4 (PSD95) on white matter in preterm infants”
Authors: Krishnan Michelle L , Van Steenwinckel Juliette , Schang Anne-Laure , Yan Jun , Arnadottir Johanna , Le Charpentier Tifenn , Csaba Zsolt , Dournaud Pascal , Cipriani Sara , Auvynet Constance , Titomanlio Luigi , Pansiot Julien , Ball Gareth , Boardman James P , Walley Andrew J , Saxena Alka , Mirza Ghazala , Fleiss Bobbi , Edwards A David , Petretto Enrico , Gressens Pierre 
Published: February 2017

Preterm birth places newborn infants in an adverse environment that leads to brain injury linked to neuroinflammation. To characterise this pathology, we present a translational bioinformatics investigation, with integration of human and mouse molecular and neuroimaging datasets to provide a deeper understanding of the role of microglia in preterm white matter damage. We examined preterm neuroinflammation in a mouse model of encephalopathy of prematurity induced by IL1B exposure, carrying out a gene network analysis of the cell-specific transcriptomic response to injury, which we extended to analysis of protein-protein interactions, transcription factors, and human brain gene expression, including translation to preterm infants by means of imaging-genetics approaches in the brain. We identified the endogenous synthesis of DLG4 (PSD95) protein by microglia in mouse and human, modulated by inflammation and development. Systemic genetic variation inDLG4was associated with structural features in the preterm infant brain, suggesting that genetic variation inDLG4may also impact white matter development and inter-individual susceptibility to injury.

Preterm birth accounts for 11% of all births1, and is the leading global cause of deaths under 5 years of age2. Over 30% of survivors experience motor and/or cognitive problems from birth3, 4, which last into adulthood5. These problems include a 3-8 fold increased risk of symptoms and disorders associated with anxiety, inattention and social and communication problems compared to term-born infants6. Prematurity is associated with a 4-12 fold increase in the prevalence of Autism Spectrum Disorders (ASD) compared to the general population7, as well as a risk ratio of 7.4 for bipolar affective disorder among infants born below 32 weeks of gestation8.

The characteristic brain injury observed in contemporary cohorts of preterm born infants includes changes to the grey and white matter tissues, that specifically include oligodendrocyte maturation arrest, hypomyelination and cortical changes visualised as decreases in fractional anisotropy9–13. Exposure of the fetus and postnatal infant to systemic inflammation is an important contributing factor to brain injury in preterm born infants12, 14, 15, and the persistence of inflammation is associated with poorer neurological outcome16. Sources of systemic inflammation include maternal/fetal infections such as chorioamnionitis (which it is estimated affects a large number of women at a sub-clinical level), with the effect of systemic inflammation in the brain being mediated predominantly by the microglial response17.

Microglia are unique yolk-sac derived resident phagocytes of the brain18, 19, found preferentially within the developing white matter as a matter of normal developmental migration12. Microglial products associated with white matter injury include pro-inflammatory cytokines, such as interleukin-1β (IL1B) and tumour necrosis factor α (TNF-α)20, which can lead to a sub-clinical inflammatory situation associated with unfavourable outcomes21. In addition to being key effector cells in brain inflammation, they are critical for normal brain development in processes such as axonal growth and synapse formation22, 23. The role of microglia in neuroinflammation is dynamic and complex, reflected in their mutable phenotypes including both pro-inflammatory and restorative functions24. Despite their important neurobiological role, the time course and nature of the microglial responses in preterm birth are currently largely unknown, and the interplay of inflammatory and developmental processes is also unclear. We, and others, believe that a better understanding of the molecular mechanisms underlying microglial function could harness their beneficial effects and mitigate the brain injury of prematurity and other states of brain inflammation25, 26

A clinically relevant experimental mouse model of IL1B-induced systemic inflammation has been developed to study the changes occurring in the preterm human brain27, 28. This model recapitulates the hallmarks of encephalopathy of prematurity including oligodendrocyte maturation delay with consequent dysmyelination, associated magnetic resonance imaging (MRI) phenotypes and behavioural deficits. Here, we take advantage of this model system to characterise the molecular underpinnings of the microglial response to IL1B-driven systemic inflammation and investigate its role in concurrent development.

In preterm infants MRI is used extensively to provide in-vivo correlates of white and grey matter pathology, allowing clinical assessment and prognostication. Diffusion MRI (d-MRI) measures the displacement of water molecules in the brain, and provides insight into the underlying tissue structure. Various d-MRI measures of white matter have been associated with developmental outcome in children born preterm29–32, with up to 60% of inter-individual variability in structural and functional features attributable to genetic factors33, 34. White matter abnormalities are linked to associated grey matter changes at both the imaging and cellular level10, 35, 36, with functional and structural consequences lasting into adulthood37, 38. Tract Based Statistics (TBSS) allows quantitative whole-brain white matter analysis of d-MRI data at the voxel level while avoiding problems due to contamination by signals arising from grey matter39. This permits voxel-wise statistical testing and inferences to be made about group differences or associations with greater statistical power. TBSS has been shown to be an effective tool for studying white matter development and injury in the preterm brain40, providing a macroscopic in vivo quantitative measure of white matter integrity that is associated with cognitive, fine motor, and gross motor outcome11, 41, 42.

In this work we take a translational systems biology approach to investigate the role of microglia in preterm neuroinflammation and brain injury. We integrate microglial cell-type specific data from a mouse model of perinatal neuroinflammatory brain injury with experimental ex vivo and in vitro validation, translation to the human brain across the lifespan including analysis of human microglia, and assessment of the impact of genetic variation on structure of the preterm brain. We add to the understanding of the neurobiology of prematurity by: a) revealing the endogenous expression of DLG4 (PSD95) by microglia in early development, which is modulated by developmental stage and inflammation; and b) finding an association between systemic genetic variability in DLG4 and white matter structure in the preterm neonatal brain.