Julie E. Simpson

White Matter Lesions in an Unselected Cohort of the Elderly Molecular Pathology Suggests Origin From Chronic Hypoperfusion Injury

Background and Purpose— “Incidental” MRI white matter (WM) lesions, comprising periventricular lesions (PVLs) and deep subcortical lesions (DSCLs), are common in the aging brain. Direct evidence of ischemia associated with incidental WM lesions (WMLs) has been lacking, and their pathogenesis is unresolved. Methods— A population-based, postmortem cohort (n=456) of donated brains was examined by MRI and pathology. In a subsample of the whole cohort, magnetic resonance images were used to sample and compare WMLs and nonlesional WM for molecular markers of hypoxic injury. Results— PVL severity was associated with loss of ventricular ependyma (P=0.004). For DSCLs, there was arteriolar sclerosis compared with normal WM (vessel wall thickness and perivascular enlargement; both P<0.001). Capillary endothelial activation (ratio of intercellular adhesion molecule to basement membrane collagen IV; P<0.001) and microglial activation (CD68 expression; P=0.002) were elevated in WMLs. Immunoreactivity for hypoxia-inducible factors (HIFs) HIF1&agr; and HIF2&agr; was elevated in DSCLs (P=0.003 and P=0.005). Other hypoxia-regulated proteins were also increased in WMLs: matrix metalloproteinase-7 (PVLs P<0.001; DSCLs P=0.009) and the number of neuroglobin-positive cells (WMLs P=0.02) reaching statistical significance. The severity of congophilic amyloid angiopathy was associated with increased HIF1&agr; expression in DSCLs (P=0.04). Conclusion— The data support a hypoxic environment within MRI WMLs. Persistent HIF expression may result from failure of normal adaptive mechanisms. WM ischemia appears to be a common feature of the aging brain.

Astrocyte phenotype in relation to Alzheimer-type pathology in the ageing brain.

Astrocyte pathology occurs in association with Alzheimers disease (AD) and in brain ageing, but is poorly characterised. We sought to define the detailed cellular pathology of astrocytes, the extent of population variation and the relationship to Alzheimer-type changes in a population-based cohort. Three staining patterns were associated with GFAP and excitatory amino acid transporter 2 (EAAT2): minimal, moderate or extensive immunoreactivity. GFAP and EAAT2 expression were inversely related (p=0.015), with trends to increased expression of GFAP (p=0.019) and decreased expression of EAAT2 (p=ns) with increasing Braak stage. GFAP and EAAT2 correlated incompletely with beta-amyloid and tau immunoreactivity. However, gliosis increased with increasing burden of neuritic (p=0.011), but not diffuse (p=ns), plaques. Double-staining revealed distinct subsets of astrocytes; GFAP(+)EAAT(-), GFAP(-)EAAT(+), or GFAP(+)EAAT(+). In contrast to the variation in GFAP and EAAT2, levels of EAAT1 and S100B showed consistent staining patterns. Alzheimer-type pathology only partially explains the variation in gliosis and astrocyte functional markers, suggesting that other factors contribute to the population variance in astrocyte pathology.

White matter lesions in an unselected cohort of the elderly : astrocytic, microglial and oligodendrocyte precursor cell responses

Hyperintense lesions are frequently identified in T2‐weighted magnetic resonance images (MRI) in the ageing brain. The pathological correlate and pathogenesis of white matter lesions (WML) remain unclear, and it is uncertain whether pathology and pathogenesis differ in periventricular lesions (PVL) compared with deep subcortical lesions (DSCL). Therefore we characterized astrocytic, microglial and oligodendrocyte responses in PVL and DSCL and compared them with control white matter using immunohistochemistry. Both PVL and DSCL were associated with severe myelin loss and increased microglia (P = 0.069 and P < 0.001), compared with nonlesional aged brain. Clasmatodendritic astroglia, immunoreactive for the serum protein fibrinogen, were present in 67% of PVL examined and 42% of DSCL. Compared with control and DSCL cases, more MAP‐2 +13 positive remyelinating oligodendrocytes (P = 0.003 and P = 0.035) and platelet‐derived growth factor α receptor positive reactive astrocytes (P < 0.001) were present in the perilesional white matter of PVL. In addition to a role for hypoperfusion, our data suggest that dysfunction of the blood–brain barrier may also contribute to the pathogenesis of a proportion of cerebral WML associated with ageing, and that attempts at remyelination are only associated with PVL and not DSCL.

MicroRNA-155 negatively affects blood–brain barrier function during neuroinflammation

Blood–brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR‐155 as a critical miRNA in neuroinflammation at the BBB. miR‐155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR‐155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR‐155 was strongly and rapidly upregulated by inflammatory cytokines. miR‐155 up‐regulation mimicked cytokine‐induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR‐155 partially prevented a cytokine‐induced increase in permeability. Furthermore, miR‐155 modulated brain endothelial barrier function by targeting not only cell–cell complex molecules such as annexin‐2 and claudin‐1, but also focal adhesion components such as DOCK‐1 and syntenin‐1. We propose that brain endothelial miR‐155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders.—Lopez‐Ramirez, M. A., Wu, D., Pryce, G., Simpson, J. E., Reijerkerk, A., King‐Robson, J., Kay, O, de Vries, H. E., Hirst, M. C., Sharrack, B., Baker D., Male, D. K., Michael, G. J., Romero, I. A. MicroRNA‐155 negatively affects blood–brain barrier function during neuroinflammation. FASEB J. 28, 2551–2565 (2014). www.fasebj.org

Microarray analysis of the astrocyte transcriptome in the aging brain: relationship to Alzheimer's pathology and APOE genotype

Astrocytes contribute to a variety of functions in the brain, including homeostasis, synapse formation, plasticity, and metabolism. Astrocyte dysfunction may disrupt their normal role, including neuronal support, thereby contributing to neurodegenerative pathologies, including Alzheimers disease (AD). To understand the role of astrocytes in the pathogenesis of age-related disorders, we isolated astrocytes by laser capture microdissection, using glial fibrillary acidic protein (GFAP) as a marker, and characterized the astrocyte transcriptome at different Braak neurofibrillary tangle stages in postmortem temporal cortex samples derived from the Medical Research Council Cognitive Function and Ageing Study (MRC CFAS) cohort, using microarray analysis. The largest number of significant, differentially expressed genes were identified when the expression profile of astrocytes from isocortical stages of neurofibrillary tangle pathology (Braak stages V-VI) were compared with entorhinal stages (Braak stages I-II). Dysregulation of genes associated with the actin cytoskeleton, proliferation, apoptosis, and ubiquitin-mediated proteolysis occurred at low Braak stages, while altered regulation of intracellular signaling pathways, including insulin, phosphatidylinositol 3-kinase (PI3K)/Akt, and mitogen-activated protein kinase (MAPK) pathways were primarily associated with high levels of Alzheimer-type pathology, and occurred at lower Braak stages in individuals with the APOEε4 allele. Our findings implicate astrocyte dysfunction in the pathogenesis of neurodegenerative pathology in the aging brain, and provide a basis for future candidate studies based on specific pathways.

Microglial activation in white matter lesions and nonlesional white matter of ageing brains.

White matter lesions (WML), a common feature in brain ageing, are classified as periventricular (PVL) or deep subcortical (DSCL), depending on their anatomical location. Microglial activation is implicated in a number of neurodegenerative diseases, but the microglial response in WML is poorly characterized and its role in pathogenesis unknown. We have characterized the microglial response in WML and control white matter using immunohistochemistry to markers of microglial activation and of proliferation. WML of brains from an unbiased population‐based autopsy cohort (Medical Research Councils Cognitive Function and Ageing Study) were identified by post mortem magnetic resonance imaging and sampled for histology. PVL contain significantly more activated microglia, expressing major histocompatibility complex (MHC) class II and the costimulatory molecules B7‐2 and CD40, than either control white matter (WM) or DSCL. Furthermore, we show that significantly more microglia express the replication licensing protein minichromosome maintenance protein 2 within PVL, suggesting this is a more proliferation‐permissive environment than DSCL. Although microglial activation occurs in both PVL and DSCL, our findings suggest a difference in pathogenesis between these lesion‐types: the ramified, activated microglia associated with PVL may reflect immune activation resulting from disruption of the blood brain barrier, while the microglia within DSCL may reflect an innate, amoeboid phagocytic phenotype. We also show that microglia in control WM from lesional cases express significantly more MHC II than control WM from nonlesional ageing brain, suggesting that WML occur in a ‘field‐effect’ of abnormal WM.

Alpha‐synuclein mRNA expression in oligodendrocytes in MSA

Multiple system atrophy (MSA) is a progressive neurodegenerative disease presenting clinically with parkinsonian, cerebellar, and autonomic features. α‐Synuclein (αsyn), encoded by the gene SNCA, is the main constituent of glial cytoplasmic inclusion (GCI) found in oligodendrocytes in MSA, but the methods of its accumulation have not been established. The aim of this study is to investigate alterations in regional and cellular SNCA mRNA expression in MSA as a possible substrate for GCI formation. Quantitative reverse transcription polymerase chain reaction (qPCR) was performed on postmortem brain samples from 15 MSA, 5 IPD, and 5 control cases to investigate regional expression in the frontal and occipital regions, dorsal putamen, pontine base, and cerebellum. For cellular expression analysis, neurons and oligodendrocytes were isolated by laser‐capture microdissection from five MSA and five control cases. SNCA mRNA expression was not significantly different between the MSA, IPD and control cases in all regions (multilevel model, P = 0.14). After adjusting for group effect, the highest expression was found in the occipital cortex while the lowest was in the putamen (multilevel model, P < 0.0001). At the cellular level, MSA oligodendrocytes expressed more SNCA than control oligodendrocytes and expression in MSA neurons was slightly lower than that in controls, however, these results did not reach statistical significance. We have demonstrated regional variations in SNCA expression, which is higher in cortical than subcortical regions. This study is the first to demonstrate SNCA mRNA expression by oligodendrocytes in human postmortem tissue using qPCR and, although not statistically significant, could suggest that this may be increased in MSA compared to controls. GLIA 2014;62:964–970

Population variation in oxidative stress and astrocyte DNA damage in relation to Alzheimer‐type pathology in the ageing brain

J. E. Simpson, P. G. Ince, L. J. Haynes, R. Theaker, C. Gelsthorpe, L. Baxter, G. Forster, G. L. Lace, P. J. Shaw, F. E. Matthews, G. M. Savva, C. Brayne and S. B. Wharton (2010) Neuropathology and Applied Neurobiology36, 25–40
Population variation in oxidative stress and astrocyte DNA damage in relation to alzheimer‐type pathology in the ageing brain

Microarray RNA Expression Analysis of Cerebral White Matter Lesions Reveals Changes in Multiple Functional Pathways

Background and Purpose— White matter lesions (WML) in brain aging are linked to dementia and depression. Ischemia contributes to their pathogenesis but other mechanisms may contribute. We used RNA microarray analysis with functional pathway grouping as an unbiased approach to investigate evidence for additional pathogenetic mechanisms. Methods— WML were identified by MRI and pathology in brains donated to the Medical Research Council Cognitive Function and Ageing Study Cognitive Function and Aging Study. RNA was extracted to compare WML with nonlesional white matter samples from cases with lesions (WM[L]), and from cases with no lesions (WM[C]) using RNA microarray and pathway analysis. Functional pathways were validated for selected genes by quantitative real-time polymerase chain reaction and immunocytochemistry. Results— We identified 8 major pathways in which multiple genes showed altered RNA transcription (immune regulation, cell cycle, apoptosis, proteolysis, ion transport, cell structure, electron transport, metabolism) among 502 genes that were differentially expressed in WML compared to WM[C]. In WM[L], 409 genes were altered involving the same pathways. Genes selected to validate this microarray data all showed the expected changes in RNA levels and immunohistochemical expression of protein. Conclusion— WML represent areas with a complex molecular phenotype. From this and previous evidence, WML may arise through tissue ischemia but may also reflect the contribution of additional factors like blood–brain barrier dysfunction. Differential expression of genes in WM[L] compared to WM[C] indicate a “field effect” in the seemingly normal surrounding white matter.

Alterations in the blood brain barrier in ageing cerebral cortex in relationship to Alzheimer-type pathology: A study in the MRC-CFAS population neuropathology cohort

Impairment of the blood brain barrier (BBB) in human brain ageing and its relationship to Alzheimer-type pathology remains poorly defined. We have investigated the BBB in temporal cortex of brain donations from a population-representative sample of 92 participants from the Medical Research Council Cognitive Function and Ageing Study (MRC CFAS), a longitudinal study with a programme of brain donation. BBB alteration was investigated by immunohistochemistry to albumin and fibrinogen and to the tight junction proteins claudin-5, zonula occludens-1 (ZO-1) and occludin. BBB leakage showed wide population-variation and increased with progression of Alzheimer-type pathology, though with considerable overlap between different levels of Alzheimer-type pathology. This was accompanied by increased mean vascular density, but not by down-regulation of tight junction proteins. ZO-1 and occludin were also expressed in glia. Mechanisms leading to BBB leakage in brain ageing remain to be defined, but the population-variation in BBB changes and its early increase in relationship to Alzheimer-type pathology progression suggest that BBB dysfunction contributes to brain ageing.