Johannes C. M. Schlachetzki

An environment-dependent transcriptional network specifies human microglia identity

Of mice and mens microglia Microglia are immune system cells that function in protecting and maintaining the brain. Gosselin et al. examined the epigenetics and RNA transcripts from single microglial cells and observed consistent profiles among samples despite differences in age, sex, and diagnosis. Mouse and human microglia demonstrated similar microglia-specific gene expression profiles, as well as a shared environmental response among microglia collected either immediately after surgery (ex vivo) or after culturing (in vitro). Interestingly, those genes exhibiting differences in expression between humans and mice or after culturing were often implicated in neurodegenerative diseases. Science, this issue p. eaal3222 Single-cell sequencing of brain microglia reveals ex vivo and in vitro differences in transcription. INTRODUCTION Microglia play essential roles in central nervous system homeostasis and influence diverse aspects of neuronal function, including refinement of synaptic networks and elaboration of neuromodulatory factors for memory and motor learning. Many lines of evidence indicate that dysregulation of microglial functions contributes to the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Emerging evidence from mouse and human studies also suggests that microglia influence neurodevelopmental and psychiatric disorders such as schizophrenia and depression. Most disease risk alleles associated with neurodegenerative diseases reside in noncoding regions of the genome, requiring the delineation of functional genomic elements in the relevant human cell types to establish mechanisms of causation. The recent observation that mouse brain environment strongly influences microglia-specific gene expression has implications for understanding pathogenic responses of microglia in diseases and disorders and modeling their phenotypes in vitro. RATIONALE Although dysregulation of microglial activity is genetically linked to neurodegenerative diseases and psychiatric disorders, no systematic evaluations of human microglia gene expression or regulatory landscapes are currently available. In addition, the extent to which mice provide suitable models for human microglia is unclear. The major goals of this study were to define the transcriptomes and DNA regulatory elements of human microglia ex vivo and in vitro in comparison to the mouse and to systematically relate these features to expression of genes associated with genome-wide association study (GWAS) risk alleles or exhibiting altered expression in neurodegenerative diseases and psychiatric disorders. RESULTS We used RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin sequencing to characterize the transcriptomes and epigenetic landscapes of human microglia isolated from surgically resected brain tissue in excess of that needed for diagnosis. Although some effects of underlying disease cannot be excluded, the overall pattern of gene expression was markedly consistent. Microglia-enriched genes were found to overlap significantly with genes exhibiting altered expression in neurodegenerative diseases and psychiatric disorders and with genes associated with a wide spectrum of disease-specific risk alleles. Human microglia gene expression was well correlated with mouse microglia gene expression, but numerous species-specific differences were also observed that included genes linked to human disease. More than half of the genes associated with noncoding GWAS risk alleles for Alzheimer’s disease are preferentially expressed in microglia. DNA recognition motifs enriched at active enhancers and expression of the corresponding lineage-determining transcription factors were very similar for human and mouse microglia. Transition of human and mouse microglia from the brain to tissue culture revealed remodeling of their respective enhancer landscapes and extensive down-regulation of genes that are induced in primitive mouse macrophages following migration into the fetal brain. Treatment of microglia in vitro with transforming growth factor β1 (TGF-β1) had relatively modest effects in maintaining the ex vivo pattern of gene expression. A significant subset of the genes up- or down-regulated in vitro exhibited altered expression in neurodegenerative diseases and psychiatric disorders. CONCLUSION These studies identify core features of human microglial transcriptomes and epigenetic landscapes. Intersection of the microglia-specific gene signature with GWAS and transcriptomic data supports roles of microglia as both responders and contributors to disease phenotypes. The identification of an environment-sensitive program of gene expression and corresponding regulatory elements enables inference of a conserved and dynamic transcription factor network that maintains microglia identity and function. The combinations of signaling factors in the brain necessary to maintain microglia phenotypes remain largely unknown. In concert, these findings will inform efforts to generate microglia-like cells in simple and complex culture systems and understand gene-environment interactions that influence homeostatic and pathogenic functions of microglia in the human brain. Brain environment specifies gene expression in microglia. Human microglia transcriptomes and enhancer landscapes were defined ex vivo following purification from surgically resected brain tissue and in vitro after transfer to a tissue culture environment. Dynamic changes in these features enabled delineation of transcription factors controlling an environment-dependent program of gene expression that overlaps with genes that are dysregulated in brain pathologies. Microglia play essential roles in central nervous system (CNS) homeostasis and influence diverse aspects of neuronal function. However, the transcriptional mechanisms that specify human microglia phenotypes are largely unknown. We examined the transcriptomes and epigenetic landscapes of human microglia isolated from surgically resected brain tissue ex vivo and after transition to an in vitro environment. Transfer to a tissue culture environment resulted in rapid and extensive down-regulation of microglia-specific genes that were induced in primitive mouse macrophages after migration into the fetal brain. Substantial subsets of these genes exhibited altered expression in neurodegenerative and behavioral diseases and were associated with noncoding risk variants. These findings reveal an environment-dependent transcriptional network specifying microglia-specific programs of gene expression and facilitate efforts to understand the roles of microglia in human brain diseases.

Unbiased and mobile gait analysis detects motor impairment in Parkinson's disease.

Motor impairments are the prerequisite for the diagnosis in Parkinsons disease (PD). The cardinal symptoms (bradykinesia, rigor, tremor, and postural instability) are used for disease staging and assessment of progression. They serve as primary outcome measures for clinical studies aiming at symptomatic and disease modifying interventions. One major caveat of clinical scores such as the Unified Parkinson Disease Rating Scale (UPDRS) or Hoehn&Yahr (H&Y) staging is its rater and time-of-assessment dependency. Thus, we aimed to objectively and automatically classify specific stages and motor signs in PD using a mobile, biosensor based Embedded Gait Analysis using Intelligent Technology (eGaIT). eGaIT consist of accelerometers and gyroscopes attached to shoes that record motion signals during standardized gait and leg function. From sensor signals 694 features were calculated and pattern recognition algorithms were applied to classify PD, H&Y stages, and motor signs correlating to the UPDRS-III motor score in a training cohort of 50 PD patients and 42 age matched controls. Classification results were confirmed in a second independent validation cohort (42 patients, 39 controls). eGaIT was able to successfully distinguish PD patients from controls with an overall classification rate of 81%. Classification accuracy increased with higher levels of motor impairment (91% for more severely affected patients) or more advanced stages of PD (91% for H&Y III patients compared to controls), supporting the PD-specific type of analysis by eGaIT. In addition, eGaIT was able to classify different H&Y stages, or different levels of motor impairment (UPDRS-III). In conclusion, eGaIT as an unbiased, mobile, and automated assessment tool is able to identify PD patients and characterize their motor impairment. It may serve as a complementary mean for the daily clinical workup and support therapeutic decisions throughout the course of the disease.

Granulocyte-colony stimulating factor is neuroprotective in a model of Parkinson's disease

We have recently shown that the hematopoietic Granulocyte‐Colony Stimulating Factor (G‐CSF) is neuroprotective in rodent stroke models, and that this action appears to be mediated via a neuronal G‐CSF receptor. Here, we report that the G‐CSF receptor is expressed in rodent dopaminergic substantia nigra neurons, suggesting that G‐CSF might be neuroprotective for dopaminergic neurons and a candidate molecule for the treatment of Parkinsons disease. Thus, we investigated protective effects of G‐CSF in 1‐methyl‐4‐phenylpyridinium (MPP+)‐challenged PC12 cells and primary neuronal midbrain cultures, as well as in the mouse 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) model of Parkinsons disease. Substantial protection was found against MPP+‐induced dopaminergic cell death in vitro. Moreover, subcutaneous application of G‐CSF at a dose of 40 μg/Kg body weight daily over 13 days rescued dopaminergic substantia nigra neurons from MPTP‐induced death in aged mice, as shown by quantification of tyrosine hydroxylase‐positive substantia nigra cells. Using HPLC, a corresponding reduction in striatal dopamine depletion after MPTP application was observed in G‐CSF‐treated mice. Thus our data suggest that G‐CSF is a novel therapeutic opportunity for the treatment of Parkinsons disease, because it is well‐tolerated and already approved for the treatment of neutropenic conditions in humans.

Microglial Activation in Alzheimers Disease

Alzheimers disease (AD) is a devastating chronic neurodegenerative disease with currently no available disease modifying treatment. In recent years, the peptide amyloid-beta has been proposed as the major pathogenic force in the development and progression of AD. Microglia, the resident immune and phagocytic cells of the brain, are known to constantly scan brain tissue and to respond to various pathological stimuli. Thus, newly formed plaque composed of A beta seem to activate and recruit microglia in AD transgenic mice. However, the role of microglia is only poorly understood in AD. Microglia may act as a double-edged sword being either detrimental or protective depending on the context. In this mini-review, we discuss the importance of microglia and its receptors in neuroinflammation and plaque clearance. A possible disease modifying role of blood-borne monocytes, which are close relatives of bone-marrow derived microglia, will also be addressed.

Oxidative stress-induced posttranslational modifications of alpha-synuclein: specific modification of alpha-synuclein by 4-hydroxy-2-nonenal increases dopaminergic toxicity.

Aggregation and neurotoxicity of misfolded alpha-synuclein (αSyn) are crucial mechanisms for progressive dopaminergic neurodegeneration associated with Parkinsons disease (PD). Posttranslational modifications (PTMs) of αSyn caused by oxidative stress, including modification by 4-hydroxy-2-nonenal (HNE-αSyn), nitration (n-αSyn), and oxidation (o-αSyn), have been implicated to promote oligomerization of αSyn. However, it is yet unclear if these PTMs lead to different types of oligomeric intermediates. Moreover, little is known about which PTM-derived αSyn species exerts toxicity to dopaminergic cells. In this study, we directly compared aggregation characteristics of HNE-αSyn, n-αSyn, and o-αSyn. Generally, all of them promoted αSyn oligomerization. Particularly, HNE-αSyn and n-αSyn were more prone to forming oligomers than unmodified αSyn. Moreover, these PTMs prevented the formation of amyloid-like fibrils, although HNE-αSyn and o-αSyn were able to generate protofibrillar structures. The cellular effects associated with distinct PTMs were studied by exposing modified αSyn to dopaminergic Lund human mesencephalic (LUHMES) neurons. The cellular toxicity of HNE-αSyn was significantly higher than other PTM species. Furthermore, we tested the toxicity of HNE-αSyn in dopaminergic LUHMES cells and other cell types with low tyrosine hydroxylase (TH) expression, and additionally analyzed the loss of TH-immunoreactive cells in HNE-αSyn-treated LUHMES cells. We observed a selective toxicity of HNE-αSyn to neurons with higher TH expression. Further mechanistic studies showed that HNE-modification apparently increased the interaction of extracellular αSyn with neurons. Moreover, exposure of differentiated LUHMES cells to HNE-αSyn triggered the production of intracellular reactive oxygen species, preceding neuronal cell death. Antioxidant treatment effectively protected cells from the damage triggered by HNE-αSyn. Our findings suggest a specific pathological effect of HNE-αSyn on dopaminergic neurons.

Pharmacological inhibition of Akt and downstream pathways modulates the expression of COX-2 and mPGES-1 in activated microglia

BackgroundMicroglia are considered a major target for modulating neuroinflammatory and neurodegenerative disease processes. Upon activation, microglia secrete inflammatory mediators that contribute to the resolution or to further enhancement of damage in the central nervous system (CNS). Therefore, it is important to study the intracellular pathways that are involved in the expression of the inflammatory mediators. Particularly, the role of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and glycogen synthase kinase-3 (GSK-3) pathways in activated microglia is unclear. Thus, in the present study we investigated the role of Akt and its downstream pathways, GSK-3 and mTOR, in lipopolysaccharide (LPS)-activated primary rat microglia by pharmacological inhibition of these pathways in regard to the expression of cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1) and to the production of prostaglandin (PG) E2 and PGD2.FindingsWe show that inhibition of Akt by the Akt inhibitor X enhanced the production of PGE2 and PGD2 without affecting the expression of COX-2, mPGES-1, mPGES-2 and cytosolic prostaglandin E synthase (cPGES). Moreover, inhibition of GSK-3 reduced the expression of both COX-2 and mPGES-1. In contrast, the mTOR inhibitor rapamycin enhanced both COX-2 and mPGES-1 immunoreactivity and the release of PGE2 and PGD2. Interestingly, NVP-BEZ235, a dual PI3K/mTOR inhibitor, enhanced COX-2 and reduced mPGES-1 immunoreactivity, albeit PGE2 and PGD2 levels were enhanced in LPS-stimulated microglia. However, this compound also increased PGE2 in non-stimulated microglia.ConclusionTaken together, we demonstrate that blockade of mTOR and/or PI3K/Akt enhances prostanoid production and that PI3K/Akt, GSK-3 and mTOR differently regulate the expression of mPGES-1 and COX-2 in activated primary microglia. Therefore, these pathways are potential targets for the development of novel strategies to modulate neuroinflammation.

Both systemic and local application of Granulocyte-colony stimulating factor (G-CSF) is neuroprotective after retinal ganglion cell axotomy

BackgroundThe hematopoietic Granulocyte-Colony Stimulating Factor (G-CSF) plays a crucial role in controlling the number of neutrophil progenitor cells. Its function is mediated via the G-CSF receptor, which was recently found to be expressed also in the central nervous system. In addition, G-CSF provided neuroprotection in models of neuronal cell death. Here we used the retinal ganglion cell (RGC) axotomy model to compare effects of local and systemic application of neuroprotective molecules.ResultsWe found that the G-CSF receptor is robustly expressed by RGCs in vivo and in vitro. We thus evaluated G-CSF as a neuroprotectant for RGCs and found a dose-dependent neuroprotective effect of G-CSF on axotomized RGCs when given subcutaneously. As stem stell mobilization had previously been discussed as a possible contributor to the neuroprotective effects of G-CSF, we compared the local treatment of RGCs by injection of G-CSF into the vitreous body with systemic delivery by subcutaneous application. Both routes of application reduced retinal ganglion cell death to a comparable extent. Moreover, G-CSF enhanced the survival of immunopurified RGCs in vitro.ConclusionWe thus show that G-CSF neuroprotection is at least partially independent of potential systemic effects and provide further evidence that the clinically applicable G-CSF could become a treatment option for both neurodegenerative diseases and glaucoma.

Norepinephrine enhances the LPS-induced expression of COX-2 and secretion of PGE2 in primary rat microglia

BackgroundRecent studies suggest an important role for neurotransmitters as modulators of inflammation. Neuroinflammatory mediators such as cytokines and molecules of the arachidonic acid pathway are generated and released by microglia. The monoamine norepinephrine reduces the production of cytokines by activated microglia in vitro. However, little is known about the effects of norepinephrine on prostanoid synthesis. In the present study, we investigate the role of norepinephrine on cyclooxygenase- (COX-)2 expression/synthesis and prostaglandin (PG)E2 production in rat primary microglia.ResultsInterestingly, norepinephrine increased COX-2 mRNA, but not protein expression. Norepinephrine strongly enhanced COX-2 expression and PGE2 production induced by lipopolysaccharide (LPS). This effect is likely to be mediated by β-adrenoreceptors, since β-, but not α-adrenoreceptor agonists produced similar results. Furthermore, β-adrenoreceptor antagonists blocked the enhancement of COX-2 levels induced by norepinephrine and β-adrenoreceptor agonists.ConclusionsConsidering that PGE2 displays different roles in neuroinflammatory and neurodegenerative disorders, norepinephrine may play an important function in the modulation of these processes in pathophysiological conditions.

Hematopoietic cytokines--on the verge of conquering neurology.

Two hematopoietic cytokines are currently gaining increasing attention within neurological research. Erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF) have long been known for their ability to induce the proliferation of certain populations of hematopoietic lineage cells. However, it has recently been found that EPO, G-CSF, and their respective receptors are also expressed in the human central nervous system (CNS) and may be an important part of the brains endogenous system of protection. Both hematopoietic cytokines have been shown to have neuroprotective potential in a variety of animal disease models both in vitro and in vivo, through the inhibition of apoptosis, induction of angiogenesis, exertion of anti-inflammatory and neurotrophic effects, as well as by the enhancement of neurogenesis. EPO and G-CSF have been extensively studied in the context of hematological disorders and have recently been successfully applied in the first clinical trials in stroke patients. Intravenous high-dose EPO therapy was associated with an improvement in the clinical outcome and preclinical studies with intravenous high-dose G-CSF therapy have clearly shown that it has considerable neuroprotective potential in the acute, as well as in the chronic phase of stroke. In this review, the current knowledge of the neuroprotective mechanisms of EPO and G-CSF is summarized with regard to in vitro and in vivo data. Focus is placed on the role of EPO in neurological disease models with an emphasis on its influence on functional outcome. New experimental results are assessed in detail and correlated with the findings of recent clinical studies.

Oligodendroglia and Myelin in Neurodegenerative Diseases: More Than Just Bystanders?

Oligodendrocytes, the myelinating cells of the central nervous system, mediate rapid action potential conduction and provide trophic support for axonal as well as neuronal maintenance. Their progenitor cell population is widely distributed in the adult brain and represents a permanent cellular reservoir for oligodendrocyte replacement and myelin plasticity. The recognition of oligodendrocytes, their progeny, and myelin as contributing factors for the pathogenesis and the progression of neurodegenerative disease has recently evolved shaping our understanding of these disorders. In the present review, we aim to highlight studies on oligodendrocytes and their progenitors in neurodegenerative diseases. We dissect oligodendroglial biology and illustrate evolutionary aspects in regard to their importance for neuronal functionality and maintenance of neuronal circuitries. After covering recent studies on oligodendroglia in different neurodegenerative diseases mainly in view of their function as myelinating cells, we focus on the alpha-synucleinopathy multiple system atrophy, a prototypical disorder with a well-defined oligodendroglial pathology.