Khalad Karram

Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats

Limited experimental access to the central nervous system (CNS) is a key problem in the study of human neural development, disease, and regeneration. We have addressed this problem by generating neural chimeras composed of human and rodent cells. Fetal human brain cells implanted into the cerebral ventricles of embryonic rats incorporate individually into all major compartments of the brain, generating widespread CNS chimerism. The human cells differentiate into neurons, astrocytes, and oligodendrocytes, which populate the host fore-, mid-, and hindbrain. These chimeras provide a unique model to study human neural cell migration and differentiation in a functional nervous system.

NG2 cells: properties, progeny and origin

The NG2 proteoglycan is a type 1-transmembrane protein expressed by a range of cell types within and outside the mammalian nervous system. NG2-expressing (NG2) cells are found in grey and white matter tracts of the developing and adult CNS and have previously been assumed to represent oligodendrocyte precursor cells: new work using transgenic mice has shown that NG2 cells generate oligodendrocytes, protoplasmic astrocytes and in some instances neurons in vivo. NG2 cells express GABAA receptors and the AMPA subtype of glutamate receptors. They make intimate contact to neurons prior to myelinating axons and also form electron-dense synaptic specialisations with axons in the cerebellum, cortex and hippocampus and with non-myelinated axons in the corpus callosum. These synaptic NG2 cells respond to neuronal release of glutamate and GABA. This neuron-glia interaction may thus regulate the differentiation and proliferation of NG2 cells. The C-terminal PDZ-binding motif of the NG2 protein binds several PDZ proteins including Mupp1, Syntenin and the Glutamate Receptor Interacting Protein (GRIP). Since GRIP can bind subunits of the AMPA receptors expressed by NG2 cells, the interaction between GRIP and NG2 may orientate the glial AMPA receptors towards sites of neuronal glutamate release. The origin, heterogeneity and function of NG2 cells as modulators of the neuronal network are important incompletely resolved questions.

Genetic Cell Ablation Reveals Clusters of Local Self-Renewing Microglia in the Mammalian Central Nervous System

During early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the organisms lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1 week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process.

Oligodendrocytes in mouse corpus callosum are coupled via gap junction channels formed by connexin47 and connexin32.

According to previously published ultrastructural studies, oligodendrocytes in white matter exhibit gap junctions with astrocytes, but not among each other, while in vitro oligodendrocytes form functional gap junctions. We have studied functional coupling among oligodendrocytes in acute slices of postnatal mouse corpus callosum. By whole‐cell patch clamp we dialyzed oligodendrocytes with biocytin, a gap junction‐permeable tracer. On average 61 cells were positive for biocytin detected by labeling with streptavidin‐Cy3. About 77% of the coupled cells stained positively for the oligodendrocyte marker protein CNPase, 9% for the astrocyte marker GFAP and 14% were negative for both CNPase and GFAP. In the latter population, the majority expressed Olig2 and some NG2, markers for oligodendrocyte precursors. Oligodendrocytes are known to express Cx47, Cx32 and Cx29, astrocytes Cx43 and Cx30. In Cx47‐deficient mice, the number of coupled cells was reduced by 80%. Deletion of Cx32 or Cx29 alone did not significantly reduce the number of coupled cells, but coupling was absent in Cx32/Cx47‐double‐deficient mice. Cx47‐ablation completely abolished coupling of oligodendrocytes to astrocytes. In Cx43‐deficient animals, oligodendrocyte‐astrocyte coupling was still present, but coupling to oligodendrocyte precursors was not observed. In Cx43/Cx30‐double deficient mice, oligodendrocyte‐to‐astrocyte coupling was almost absent. Uncoupled oligodendrocytes showed a higher input resistance. We conclude that oligodendrocytes in white matter form a functional syncytium predominantly among each other dependent on Cx47 and Cx32 expression, while astrocytic connexins expression can promote the size of this network.

Primary oligodendrocyte death does not elicit anti-CNS immunity.

Anti-myelin immunity is commonly thought to drive multiple sclerosis, yet the initial trigger of this autoreactivity remains elusive. One of the proposed factors for initiating this disease is the primary death of oligodendrocytes. To specifically test such oligodendrocyte death as a trigger for anti-CNS immunity, we inducibly killed oligodendrocytes in an in vivo mouse model. Strong microglia-macrophage activation followed oligodendrocyte death, and myelin components in draining lymph nodes made CNS antigens available to lymphocytes. However, even conditions favoring autoimmunity—bystander activation, removal of regulatory T cells, presence of myelin-reactive T cells and application of demyelinating antibodies—did not result in the development of CNS inflammation after oligodendrocyte death. In addition, this lack of reactivity was not mediated by enhanced myelin-specific tolerance. Thus, in contrast with previously reported impairments of oligodendrocyte physiology, diffuse oligodendrocyte death alone or in conjunction with immune activation does not trigger anti-CNS immunity.

NG2‐expressing cells in the nervous system revealed by the NG2‐EYFP‐knockin mouse

The NG2 glycoprotein is a type I membrane protein expressed by immature cells in the developing and adult mouse. NG2+ cells of the embryonic and adult brain have been principally viewed as oligodendrocyte precursor cells but have additionally been considered a fourth glial class. They are likely to be a heterogeneous population. In order to facilitate studies on the function of NG2+ cells and to characterize these cells in situ, we generated an enhanced yellow fluorescent protein (EYFP) “knockin mouse.” EYFP‐expressing cells in heterozygous knockin mice expressed the NG2 protein in all regions and at all ages studied. The EYFP+ cells did not express markers of mature glia, developing or mature neurons or microglia, but expressed markers typical for immature oligodendrocyte‐lineage cells. Examination of the hippocampus showed heterogeneity in the population with regard to expression of S100ß and glutamine synthetase. Furthermore, different subpopulations of NG2+ cells in the hippocampus could be recognized by their electrophysiological properties. genesis 46:743–757, 2008.

An Alternative Pathway of Imiquimod-Induced Psoriasis-Like Skin Inflammation in the Absence of Interleukin-17 Receptor A Signaling

Topical application of imiquimod (IMQ) on the skin of mice induces inflammation with common features found in psoriatic skin. Recently, it was postulated that IL-17 has an important role both in psoriasis and in the IMQ model. To further investigate the impact of IL-17RA signaling in psoriasis, we generated IL-17 receptor A (IL-17RA)-deficient mice (IL-17RA(del)) and challenged these mice with IMQ. Interestingly, the disease was only partially reduced and delayed but not abolished when compared with controls. In the absence of IL-17RA, we found persisting signs of inflammation such as neutrophil and macrophage infiltration within the skin. Surprisingly, already in the naive state, the skin of IL-17RA(del) mice contained significantly elevated numbers of Th17- and IL-17-producing γδ T cells, assuming that IL-17RA signaling regulates the population size of Th17 and γδ T cells. Upon IMQ treatment of IL-17RA(del) mice, these cells secreted elevated amounts of tumor necrosis factor-α, IL-6, and IL-22, accompanied by increased levels of the chemokine CXCL2, suggesting an alternative pathway of neutrophil and macrophage skin infiltration. Hence, our findings have major implications in the potential long-term treatment of psoriasis by IL-17-targeting drugs.

Functional network integration of embryonic stem cell-derived astrocytes in hippocampal slice cultures

Embryonic stem (ES) cells provide attractive prospects for neural transplantation. So far, grafting strategies in the CNS have focused mainly on neuronal replacement. Employing a slice culture model, we found that ES cell-derived glial precursors (ESGPs) possess a remarkable capacity to integrate into the host glial network. Following deposition on the surface of hippocampal slices, ESGPs actively migrate into the recipient tissue and establish extensive cell-cell contacts with recipient glia. Gap junction-mediated coupling between donor and host astrocytes permits widespread delivery of dye from single donor cells. During maturation, engrafted donor cells display morphological, immunochemical and electrophysiological properties that are characteristic of differentiating native glia. Our findings provide the first evidence of functional integration of grafted astrocytes, and depict glial network integration as a potential route for widespread transcellular delivery of small molecules to the CNS.

Novel NG2-CreERT2 knock-in mice demonstrate heterogeneous differentiation potential of NG2 glia during development

NG2 (nerve/glia antigen‐2) is a type I transmembrane glycoprotein and also known as chondroitin sulfate proteoglycan 4. In the parenchyma of the central nervous system, NG2‐expressing (NG2+) cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes (OLs) in the developing white matter. However, the differentiation potential of NG2 glia remained controversial, largely attributable to shortcomings of transgenic mouse models used for fate mapping. To minimize these restrictions and to more faithfully mimic the endogenous NG2 expression in vivo, we generated a mouse line in which the open reading frame of the tamoxifen‐inducible form of the Cre DNA recombinase (CreERT2) was inserted into the NG2 locus by homologous recombination. Results from this novel mouse line demonstrate that at different developmental stages of the brain, NG2+ cells either stayed as NG2 glia or differentiated into OLs during the whole life span. Interestingly, when Cre activity was induced at embryonic stages, a significant number of reporter+ astrocytes could be detected in the gray matter after birth. However, in other brain regions, such as olfactory bulb, brain stem, and cerebellum, all of the NG2 glia was restricted to the OL lineage. In addition, tamoxifen‐sensitive and NG2 gene locus‐dependent gene recombination could be detected in a small, but persistent population of cortical NeuN+ neurons starting from the second postnatal week. GLIA 2014;62:896–913

Gray Matter NG2 Cells Display Multiple Ca2+-Signaling Pathways and Highly Motile Processes

NG2 cells, the fourth type of glia in the mammalian CNS, receive synaptic input from neurons. The function of this innervation is unknown yet. Postsynaptic changes in intracellular Ca2+-concentration ([Ca2+]i) might be a possible consequence. We employed transgenic mice with fluorescently labeled NG2 cells to address this issue. To identify Ca2+-signaling pathways we combined patch-clamp recordings, Ca2+-imaging, mRNA-transcript analysis and focal pressure-application of various substances to identified NG2-cells in acute hippocampal slices. We show that activation of voltage-gated Ca2+-channels, Ca2+-permeable AMPA-receptors, and group I metabotropic glutamate-receptors provoke [Ca2+]i-elevations in NG2 cells. The Ca2+-influx is amplified by Ca2+-induced Ca2+-release. Minimal electrical stimulation of presynaptic neurons caused postsynaptic currents but no somatic [Ca2+]i elevations, suggesting that [Ca2+]i elevations in NG2 cells might be restricted to their processes. Local Ca2+-signaling might provoke transmitter release or changes in cell motility. To identify structural prerequisites for such a scenario, we used electron microscopy, immunostaining, mRNA-transcript analysis, and time lapse imaging. We found that NG2 cells form symmetric and asymmetric synapses with presynaptic neurons and show immunoreactivity for vesicular glutamate transporter 1. The processes are actin-based, contain ezrin but not glial filaments, microtubules or endoplasmic reticulum. Furthermore, we demonstrate that NG2 cell processes in situ are highly motile. Our findings demonstrate that gray matter NG2 cells are endowed with the cellular machinery for two-way communication with neighboring cells.