David R. Serwanski

Synaptic and nonsynaptic localization of GABAA receptors containing the α5 subunit in the rat brain

The α5 subunit of the GABAA receptors (GABAARs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of α5 subunit‐containing GABAARs (α5‐GABAARs) both in culture and in the intact brain. A large pool of α5‐GABAARs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the α5‐GABAARs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of α5‐GABAARs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic α5‐GABAARs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex. J. Comp. Neurol. 499:458–470, 2006.

The brefeldin A-inhibited GDP/GTP exchange factor 2, a protein involved in vesicular trafficking, interacts with the β subunits of the GABAA receptors

We have found that the brefeldin A‐inhibited GDP/GTP exchange factor 2 (BIG2) interacts with the β subunits of the γ‐aminobutyric acid type‐A receptor (GABAAR). BIG2 is a Sec7 domain‐containing guanine nucleotide exchange factor known to be involved in vesicular and protein trafficking. The interaction between the 110 amino acid C‐terminal fragment of BIG2 and the large intracellular loop of the GABAAR β subunits was revealed with a yeast two‐hybrid assay. The native BIG2 and GABAARs interact in the brain since both coprecipitated from detergent extracts with either anti‐GABAAR or anti‐BIG2 antibodies. In transfected human embryonic kidney cell line 293 cells, BIG2 promotes the exit of GABAARs from endoplasmic reticulum. Double label immunofluorescence of cultured hippocampal neurons and electron microscopy immunocytochemistry of rat brain tissue show that BIG2 concentrates in the trans‐Golgi network. BIG2 is also present in vesicle‐like structures in the dendritic cytoplasm, sometimes colocalizing with GABAARs. BIG2 is present in both inhibitory GABAergic synapses that contain GABAARs and in asymmetric excitatory synapses. The results are consistent with the hypotheses that the interaction of BIG2 with the GABAAR β subunits plays a role in the exocytosis and trafficking of assembled GABAAR to the cell surface.

NG2 Cells Are Distinct From Neurogenic Cells in the Postnatal Mouse Subventricular Zone

NG2 cells express the chondroitin sulfate proteoglycan NG2 and are a fourth type of glia distinct from astrocytes, oligodendrocytes, and microglia. NG2 cells generate oligodendrocytes but have also been reported to represent neuronal progenitor cells in the postnatal mouse subventricular zone (SVZ). We performed a detailed immunohistochemical analysis of NG2 cells in the mouse SVZ, rostral migratory stream (RMS), and olfactory bulb granule cell layer (OB GCL), which constitute a neurogenic niche in the postnatal forebrain. NG2 cells in the SVZ and RMS expressed the oligodendrocyte precursor cell antigen platelet‐derived growth factor receptor‐α but did not express antigens known to be expressed by neuronogenic cells in the SVZ, such as doublecortin, PSA‐NCAM, beta‐tubulin, Dlx2, or GFAP. More than 99.5% of the proliferating cells in the SVZ were NG2 negative. In the olfactory bulb, NG2 cells were found to generate primarily oligodendrocytes and a small number of astrocytes but not neurons. In the SVZ and RMS, NG2 cells were sparse and made up a much smaller fraction of the cells compared with the surrounding nonneurogenic parenchyma. Parenchymal NG2 cells were often located along the border of the SVZ and RMS. The abundance of NG2 cells increased in the distal parts of the RMS and especially in the OB GCL, where NG2 cell processes were seen in close proximity to many maturing interneurons. Our findings indicate that NG2 cells do not represent neuronal progenitor cells in the postnatal SVZ but are likely to be oligodendrocyte precursor cells. J. Comp. Neurol. 512:702–716, 2009.

Astrocytic Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes Oligodendrocyte Differentiation and Enhances CNS Myelination

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an extracellular protein and endogenous regulator of matrix metalloproteinases (MMPs) secreted by astrocytes in response to CNS myelin injury. We have previously reported that adult TIMP-1 knock-out (KO) mice exhibit poor myelin repair following demyelinating injury. This observation led us to hypothesize a role for TIMP-1 in oligodendrogenesis and CNS myelination. Herein, we demonstrate that compact myelin formation is significantly delayed in TIMP-1 KO mice, a situation that coincided with dramatically reduced numbers of white matter astrocytes in the developing CNS. Analysis of differentiation in CNS progenitor cells (neurosphere) cultures from TIMP-1 KO mice revealed a specific deficit of NG2+ oligodendrocyte progenitor cells. Application of recombinant murine TIMP-1 (rmTIMP-1) to TIMP-1 KO neurosphere cultures evoked a dose-dependent increase in NG2+ cell numbers, while treatment with GM6001, a potent broad-spectrum MMP inhibitor did not. Similarly, administration of rmTIMP-1 to A2B5+ immunopanned oligodendrocyte progenitors significantly increased the number of differentiated O1+ oligodendrocytes, while antisera to TIMP-1 reduced oligodendrocyte numbers. We also determined that A2B5+ oligodendrocyte progenitors grown in conditioned media derived from TIMP-1 KO primary glial cultures resulted in reduced differentiation of mature O1+ oligodendrocytes. Finally, we report that addition of rmTIMP-1 to primary glial cultures resulted in a dose-dependent proliferative response of astrocytes. Together, these findings describe a previously uncharacterized role for TIMP-1 in the regulation of oligodendrocytes and astrocytes during development and provide a novel function for TIMP-1 on myelination in the developing CNS.

Olig2-dependent developmental fate switch of NG2 cells.

NG2-expressing cells (NG2 cells or polydendrocytes) generate oligodendrocytes throughout the CNS and a subpopulation of protoplasmic astrocytes in the gray matter of the ventral forebrain. The mechanisms that regulate their oligodendrocyte or astrocyte fate and the degree to which they exhibit lineage plasticity in vivo have remained unclear. The basic helix-loop-helix transcription factor Olig2 is required for oligodendrocyte specification and differentiation. We have found that Olig2 expression is spontaneously downregulated in NG2 cells in the normal embryonic ventral forebrain as they differentiate into astrocytes. To further examine the role of Olig2 in NG2 cell fate determination, we used genetic fate mapping of NG2 cells in constitutive and tamoxifen-inducible Olig2 conditional knockout mice in which Olig2 was deleted specifically in NG2 cells. Constitutive deletion of Olig2 in NG2 cells in the neocortex and corpus callosum but not in ventral forebrain caused them to convert their fate into astrocytes, with a concomitant severe reduction in the number of oligodendrocytes and myelin. Deletion of Olig2 in NG2 cells in perinatal mice also resulted in astrocyte generation from neocortical NG2 cells. These observations indicate that the developmental fate of NG2 cells can be switched by altering a single transcription factor Olig2.

Platelet-derived growth factor receptors differentially inform intertumoral and intratumoral heterogeneity

Growth factor-mediated proliferation and self-renewal maintain tissue-specific stem cells and are frequently dysregulated in cancers. Platelet-derived growth factor (PDGF) ligands and receptors (PDGFRs) are commonly overexpressed in gliomas and initiate tumors, as proven in genetically engineered models. While PDGFRα alterations inform intertumoral heterogeneity toward a proneural glioblastoma (GBM) subtype, we interrogated the role of PDGFRs in intratumoral GBM heterogeneity. We found that PDGFRα is expressed only in a subset of GBMs, while PDGFRβ is more commonly expressed in tumors but is preferentially expressed by self-renewing tumorigenic GBM stem cells (GSCs). Genetic or pharmacological targeting of PDGFRβ (but not PDGFRα) attenuated GSC self-renewal, survival, tumor growth, and invasion. PDGFRβ inhibition decreased activation of the cancer stem cell signaling node STAT3, while constitutively active STAT3 rescued the loss of GSC self-renewal caused by PDGFRβ targeting. In silico survival analysis demonstrated that PDGFRB informed poor prognosis, while PDGFRA was a positive prognostic factor. Our results may explain mixed clinical responses of anti-PDGFR-based approaches and suggest the need for integration of models of cancer as an organ system into development of cancer therapies.

Transcellular transport of CCL2 across brain microvascular endothelial cells.

The means by which the chemokine CCL2 produced in the brain parenchyma can recruit leukocytes lying behind the highly impervious endothelium of the blood–brain barrier (BBB) has remained a paradox. As other chemokines have been evidenced to stimulate their own synthesis and release by peripheral microvascular endothelial cells, and/or undergo transcytosis in the abluminal‐to‐luminal direction, we determined whether CCL2 experiences similar fates across brain microvascular endothelial cells (BMEC). Using cultured BMEC as a paradigm of the BBB, it was observed that exogenous unlabeled CCL2 actually depressed the release of endogenous CCL2, and further caused diminished CCL2 mRNA levels in these cells. On the other hand, exogenous 125I‐labeled CCL2 exhibited transport across BMEC in a manner that was sensitive to temperature, competition by excess unlabeled CCL2 but not unlabeled CCL3, knockdown of caveolin‐1/caveolae, and elimination of the cognate CCL2 receptor CCR2. These results implied a facet of CCL2 transport by a transcellular mechanism partly involving binding of CCL2 to CCR2, and subsequent transfer to caveolae vesicles for transcytosis. This notion was supported by double‐label immuno‐electronmicroscopy, which revealed co‐localization of caveolin‐1 with exogenous CCL2, during this chemokine’s transit across BMEC. Collectively, these findings provide a rationale by which CCL2, deposited on the abluminal side of the brain microvasculature during inflammatory episodes, can be relayed across the BBB to foster leukocyte recruitment.

NG2 cells are not a major source of reactive astrocytes after neocortical stab wound injury

NG2 cells are an abundant glial cell type in the adult brain. They are distinct from astrocytes, mature oligodendrocytes, and microglia. NG2 cells generate oligodendrocytes and a subpopulation of protoplasmic astrocytes in the ventral forebrain during development. To determine whether NG2 cells generate reactive astrocytes in the lesioned brain, stab wound injury was created in adult NG2creBAC:ZEG double transgenic mice, in which enhanced green fluorescent protein (EGFP) is expressed in NG2 cells and their progeny, and the phenotype of the EGFP+ cells was analyzed at 10 and 30 days post lesion (dpl). The majority (>90%) of the reactive astrocytes surrounding the lesion that expressed glial fibrillary acidic protein (GFAP) lacked EGFP expression, and conversely the majority (>90%) of EGFP+ cells were GFAP‐negative. However, 8% of EGFP+ cells co‐expressed GFAP at 10 dpl. Most of these EGFP+GFAP+ cells were morphologically distinct from hypertrophic reactive astrocytes and exhibited weak GFAP expression. NG2 was detected in a fraction of the EGFP+GFAP+ cells found at 10 dpl. By 30 dpl the number of EGFP+GFAP+ cells had decreased more than four‐fold from 10 dpl. A similar transient appearance of EGFP+GFAP+ cells with simple morphology was observed in NG2creER™:ZEG double transgenic mice in which EGFP expression had been induced in NG2 cells prior to injury. NG2 cell‐specific deletion of the oligodendrocyte lineage transcription factor Olig2 using NG2creER™:Olig2fl/fl:ZEG triple transgenic mice did not increase the number of EGFP+ reactive astrocytes. These findings suggest that NG2 cells are not a major source of reactive astrocytes in the neocortex.

GRIP1 in GABAergic synapses.

The glutamate receptor‐interacting protein GRIP1 is present in glutamatergic synapses and interacts with the GluR2/3/4c subunits of the AMPA receptors. This interaction plays important roles in trafficking, synaptic targeting, and recycling of AMPA receptors as well as in the plasticity of glutamatergic synapses. Although GRIP1 has been shown to be present at GABAergic synapses in cultured neurons, the use of EM (electron microscopy) immunocytochemistry in the intact brain has failed to convincingly reveal the presence of GRIP1 in GABAergic synapses. Therefore, most studies on GRIP1 have focused on glutamatergic synapses. By using mild tissue fixation and embedding in EM, we show that in the intact brain the 7‐PDZ domain GRIP1a/b is present not only in glutamatergic synapses but also in GABAergic synapses. In GABAergic synapses GRIP1a/b localizes both at the presynaptic terminals and postsynaptically, being frequently localized on the synaptic membranes or the synaptic junctional complex. Considerably higher density of GRIP1a/b is found in the presynaptic GABAergic terminals than in the glutamatergic terminals, while the density of GRIP1a/b in the postsynaptic complex is similar in both types of synapses. The results also show that the 7‐PDZ and the shorter 4‐PDZ domain splice forms of GRIP1 (GRIP1c 4‐7) frequently colocalize with each other in individual GABAergic and glutamatergic synapses. The results suggest that GRIP1 splice forms might play important roles in brain GABAergic synapses. J. Comp. Neurol. 488:11–27, 2005.

Two pools of Triton X‐100‐insoluble GABAA receptors are present in the brain, one associated to lipid rafts and another one to the post‐synaptic GABAergic complex

Rat forebrain synaptosomes were extracted with Triton X‐100 at 4°C and the insoluble material, which is enriched in post‐synaptic densities (PSDs), was subjected to sedimentation on a continuous sucrose gradient. Two pools of Triton X‐100‐insoluble γ‐aminobutyric acid type‐A receptors (GABAARs) were identified: (i) a higher‐density pool (ρ = 1.10–1.15 mg/mL) of GABAARs that contains the γ2 subunit (plus α and β subunits) and that is associated to gephyrin and the GABAergic post‐synaptic complex and (ii) a lower‐density pool (ρ = 1.06–1.09 mg/mL) of GABAARs associated to detergent‐resistant membranes (DRMs) that contain α and β subunits but not the γ2 subunit. Some of these GABAARs contain the δ subunit. Two pools of GABAARs insoluble in Triton X‐100 at 4°C were also identified in cultured hippocampal neurons: (i) a GABAAR pool that forms clusters that co‐localize with gephyrin and remains Triton X‐100‐insoluble after cholesterol depletion and (ii) a GABAAR pool that is diffusely distributed at the neuronal surface that can be induced to form GABAAR clusters by capping with an anti‐α1 GABAAR subunit antibody and that becomes solubilized in Triton X‐100 at 4°C after cholesterol depletion. Thus, there is a pool of GABAARs associated to lipid rafts that is non‐synaptic and that has a subunit composition different from that of the synaptic GABAARs. Some of the lipid raft‐associated GABAARs might be involved in tonic inhibition.