Neuronal CC chemokines:the distinct roles of CCL21 and CCL2 in neuropathic pain

The development of neuropathic pain in response to peripheral nerve lesion for a large part depends on microglia located at the dorsal horn of the spinal cord. Thus the injured nerve initiates a response of microglia, which represents the start of a cascade of events that leads to neuropathic pain development. For long it remained obscure how a nerve injury in the periphery would initiate a microglia response in the dorsal horn of the spinal cord. Recently, two chemokines have been suggested as potential factors that mediate the communication between injured neurons and microglia namely CCL2 and CCL21. This assumption is based on the following findings. Both chemokines are not found in healthy neurons, but are expressed in response to neuronal injury. In injured dorsal root ganglion cells CCL2 and CCL21 are expressed in vesicles in the soma and transported through the axons of the dorsal root into the dorsal horn of the spinal cord. Finally, microglia in vitro are known to respond to CCL2 and CCL21. Whereas the microglial chemokine receptor involved in CCL21-induced neuropathic pain is not yet defined the situation concerning the receptors for CCL2 in microglia in vivo is even less clear. Recent results obtained in transgenic animals clearly show that microglia in vivo do not express CCR2 but that peripheral myeloid cells and neurons do. This suggests that CCL2 expressed by injured dorsal root neurons does not act as neuron-microglia signal in contrast to CCL21. Instead, CCL2 in the injured dorsal root ganglia (DRG) may act as autocrine or paracrine signal and may stimulate first or second order neurons in the pain cascade and/or attract CCR2-expressing peripheral monocytes/macrophages to the spinal cord

Insulin-Like Growth Factor-1 Is Neuroprotective in Aged Rats With Ischemic Stroke

Post-stroke systemic injections of insulin-like growth factor-1 (IGF-1) exert neuroprotective effects in rats. In the current study, we aimed to test the efficacy of IGF-1 neuroprotection in aged rats (24-25 months old) and to compare the results with adult rats (6-7 months old). Furthermore, we addressed putative differences in microglial responses to IGF-1 in adult and aged rats. Rats were subjected to ischemic stroke while they were conscious by infusing endothelin-1 (Et-1) through a guide cannula that was implemented in the vicinity of the middle cerebral artery (MCA). Rats were given subcutaneous injections of IGF-1 (1 mg/kg) at 30 min and 120 min after the insult. Post-stroke IGF-1 treatment reduced the infarct size by 34% and 38% in aged and adult rats, respectively. The IGF-1 treated adult rats also showed significant improvement in sensorimotor function following stroke, while this function was not significantly affected in aged rats. Furthermore, aged rats displayed exaggerated activation of microglia in the ischemic hemisphere. Significant reduction of microglial activation by IGF-1 was only detected at specific regions in the ipsilateral hemisphere of adult rats. We show that IGF-1 reduced infarct size in aged rats with an ischemic stroke. It remains to be established, however, whether the age-related changes in microglial function affect the improvement in behavioral outcomes

Mouse models of central nervous system ageing

Ageing is accompanied by decreased overall fitness and performance. Studying brain ageing in humans is challenging due to limited or no access to healthy tissue, limited opportunities for interventions and complicated confounding factors. The generation of mouse ageing models with uniform genetic backgrounds significantly contributed to understanding (brain) ageing at the molecular level. Research has focused on evolutionarily conserved mechanisms or pathways that control ageing to facilitate data extrapolation to humans. Understanding how these pathways contribute to pathological ageing may help us understand human central nervous system (CNS) ageing and assist in the development of possible therapeutic targets. In this review, we focus on the functional consequences and pathological changes in the CNS of ageing mouse models

Neuroprotection by Insulin-like Growth Factor-1 in Rats with Ischemic Stroke is Associated with Microglial Changes and a Reduction in Neuroinflammation

We and others have shown that insulin-like growth factor-1 (IGF-1) is neuroprotective when administered systemically shortly following stroke. In the current study, we addressed the hypothesis that microglia mediate neuroprotection by IGF-1 following ischemic stroke. Furthermore, we investigated whether IGF-1 modulates pro- and anti-inflammatory mediators in ischemic brain with a special reference to microglia. Ischemic stroke was induced in normal conscious Wistar rats by infusing the vasoconstrictor, endothelin-1 (Et-1), next to middle cerebral artery (MCA). IGF-1 (300 μg) was injected subcutaneously (SC) at 30 and 120 min following stroke. Microglial inhibitor, minocycline, was injected intraperitoneally (IP) at 1 h before stroke (25 mg/kg) and 11 h after stroke (45 mg/kg). Post-stroke IGF-1 treatment reduced the infarct size and increased the sensorimotor function which coincided with an increase in the number of ameboid microglia in the ischemic cortex. Minocycline treatment abrogated the increase in ameboid microglia by IGF-1, while the effect of IGF-1 in the reduction of infarct size was only partially affected. IGF-1 suppressed mRNA expression of inducible nitric oxide synthase (iNOS) and interleukin (IL)-1β in the ischemic hemisphere, while in purified microglia, only iNOS expression levels were reduced. Our findings show that microglia are a target for IGF-1 and that neuroprotection by IGF-1 coincides with down-regulation of inflammatory mediators which could be instrumental to the beneficial effects

Transcriptional profiling of microglia; current state of the art and future perspectives

Microglia are the tissue macrophages of the central nervous system (CNS) and the first to respond to CNS dysfunction and disease. Gene expression profiling of microglia during development, under homeostatic conditions, and in the diseased CNS provided insight in microglia functions and changes thereof. Single-cell sequencing studies further contributed to our understanding of microglia heterogeneity in relation to age, sex, and CNS disease. Recently, single nucleus gene expression profiling was performed on (frozen) CNS tissue. Transcriptomic profiling of CNS tissues by (single) nucleus RNA-sequencing has the advantage that it can be applied to archived and well-stratified frozen specimens. Here, we give an overview of the significant advances recently made in microglia transcriptional profiling. In addition, we present matched cellular and nuclear microglia RNA-seq datasets we generated from mouse and human CNS tissue to compare cellular versus nuclear transcriptomes from fresh and frozen samples. We demonstrate that microglia can be similarly profiled with cell and nucleus profiling, and importantly also with nuclei isolated from frozen tissue. Nuclear microglia transcriptomes are a reliable proxy for cellular transcriptomes. Importantly, lipopolysaccharide-induced changes in gene expression were conserved in the nuclear transcriptome. In addition, heterogeneity in microglia observed in fresh samples was similarly detected in frozen nuclei of the same donor. Together, these results show that microglia nuclear RNAs obtained from frozen CNS tissue are a reliable proxy for microglia gene expression and cellular heterogeneity and may prove an effective strategy to study of the role of microglia in neuropathology

Linalool attenuates oxidative stress and mitochondrial dysfunction mediated by glutamate and NMDA toxicity

Mitochondrial dysfunction and inflammation contribute to the initiation and development of several brain pathological conditions, including Alzheimer's disease and cerebral ischemia. Linalool is an aromatic plant-derived monoterpene alcohol with reported anti-inflammatory, and anti-oxidant properties. We investigated the role of linalool on glutamate-induced mitochondrial oxidative stress in immortalized neuronal HT-22 cells. Glutamate induced oxidative stress in neuronal cells, as detected by real-time cell impedance measurements, MTT assay, and analysis of Annexin V/PI. Administration of linalool 100 μM reduced cell death mediated by glutamate. Staining of glutamate-stimulated mitochondria by MitoTracker revealed improved morphology in the presence of linalool. Furthermore, we demonstrated a potential neuroprotective effect of linalool in conditions of oxidative stress by a reduction of mitochondrial ROS and mitochondrial calcium levels, and by preserving mitochondrial membrane potential. Experiments using both high-resolution respirometry and Seahorse Extracellular flux analyzer showed that linalool was able to promote an increase in uncoupled respiration that could contribute to its neuroprotective capacity. Linalool protection was validated using organotypic hippocampal slices as ex vivo model with NMDA as a stimulus to induce excitotoxity cell damage. These results demonstrate that linalool is protective in an in vitro model of glutamate-induced oxidative stress and in an ex-vivo model for excitotoxity, proposing linalool as a potential therapeutic agent against neurodegenerative brain diseases where oxidative stress contributes to the pathology of the disease

Microglia morphotyping in the adult mouse CNS using hierarchical clustering on principal components reveals regional heterogeneity but no sexual dimorphism

Microglia are the resident macrophages of the central nervous system (CNS) and play a pivotal role in immune surveillance and CNS homeostasis. Morphological transitions in microglia are indicative for local changes in the CNS microenvironment and serve as a proxy for the detection of alterations in the CNS, both in health and disease. Current strategies to ‘measure’ microglia combine advanced morphometrics with clustering approaches to identify and categorize microglia morphologies. However, these studies are labor intensive and clustering approaches are often subject to relevant feature selection bias. Here, we provide a morphometrics pipeline with user-friendly computational tools for image segmentation, automated feature extraction and morphological categorization of microglia by means of hierarchical clustering on principal components (HCPC) without the need for feature inclusion criteria. With this pipeline we provide new and detailed insights in the distribution of microglia morphotypes across sixteen CNS regions along the rostro-caudal axis of the adult C57BL/6J mouse CNS. Although regional variations in microglia morphologies were evident, we found no evidence for male–female dimorphism at any CNS region investigated, indicating that - by and large - microglia in adult male and female mice are morphometrically indistinguishable. Taken together, our newly developed pipeline provides valuable tools for objective and unbiased identification and categorization of microglia morphotypes and can be applied to any CNS (disease) model.</p

Overexpression of Polysialylated Neural Cell Adhesion Molecule Improves the Migration Capacity of Induced Pluripotent Stem Cell-Derived Oligodendrocyte Precursors

Cell replacement therapy aiming at the compensation of lost oligodendrocytes and restoration of myelination in acquired or congenital demyelination disorders has gained considerable interest since the discovery of induced pluripotent stem cells (iPSCs). Patient-derived iPSCs provide an inexhaustible source for transplantable autologous oligodendrocyte precursors (OPCs). The first transplantation studies in animal models for demyelination with iPSC-derived OPCs demonstrated their survival and remyelinating capacity, but also revealed their limited migration capacity. In the present study, we induced overexpression of the polysialylating enzyme sialyltransferase X (STX) in iPSC-derived OPCs to stimulate the production of polysialic acid-neuronal cell adhesion molecules (PSA-NCAMs), known to promote and facilitate the migration of OPCs. The STX-overexpressing iPSC-derived OPCs showed a normal differentiation and maturation pattern and were able to downregulate PSA-NCAMs when they became myelin-forming oligodendrocytes. After implantation in the demyelinated corpus callosum of cuprizone-fed mice, STX-expressing iPSC-derived OPCs demonstrated a significant increase in migration along the axons. Our findings suggest that the reach and efficacy of iPSC-derived OPC transplantation can be improved by stimulating the OPC migration potential via specific gene modulation

The Kaleidoscope of Microglial Phenotypes

Gene expression analyses of microglia, the tissue-resident macrophages of the central nervous system (CNS), led to the identification of homeostatic as well as neurological disease-specific gene signatures of microglial phenotypes. Upon alterations in the neural microenvironment, either caused by local insults from within the CNS (during neurodegenerative diseases) or by macroenvironmental incidents, such as social stress, microglia can switch phenotypes-generally referred to as "microglial activation." The interplay between the microenvironment and its influence on microglial phenotypes, regulated by (epi) genetic mechanisms, can be imagined as the different colorful crystal formations (microglial phenotypes) that change upon rotation (microenvironmental changes) of a kaleidoscope. In this review, we will discuss microglial phenotypes in relation to neurodevelopment, homeostasis, in vitro conditions, aging, and neurodegenerative diseases based on transcriptome studies. By overlaying these disease-specific microglial signatures, recent publications have identified a specific set of genes that is differentially expressed in all investigated diseases, called a microglial core gene signature with multiple diseases. We will conclude this review with a discussion about the complexity of this microglial core gene signature associated with multiple diseases

Regionally diverse astrocyte subtypes and their heterogeneous response to EAE

Astrocytes fulfil many functions in the central nervous system (CNS), including contribution to the blood brain barrier, synapse formation, and trophic support. In addition, they can mount an inflammatory response and are heterogeneous in morphology and function. To extensively characterize astrocyte subtypes, we FACS-isolated and gene expression profiled distinct astrocyte subtypes from three central nervous system regions; forebrain, hindbrain and spinal cord. Astrocyte subpopulations were separated based on GLAST/SLC1A3 and ACSA-2/ATP1B2 cell surface expression. The local brain environment proved key in establishing different transcriptional programs in astrocyte subtypes. Transcriptional differences between subtypes were also apparent in experimental autoimmune encephalomyelitis (EAE) mice, where these astrocyte subtypes showed distinct responses. While gene expression signatures associated with blood-brain barrier maintenance were lost, signatures involved in neuroinflammation and neurotoxicity were increased in spinal cord astrocytes, especially during acute disease stages. In chronic stages of EAE, this reactive astrocyte signature was slightly decreased, while obtaining a more proliferative profile, which might be relevant for glia scar formation and tissue regeneration. Morphological heterogeneity of astrocytes previously indicated the presence of astrocyte subtypes, and here we show diversity based on transcriptome variation associated with brain regions and differential responsiveness to a neuroinflammatory insult (EAE)