Combinational Spinal GAD65 Gene Delivery and Systemic GABA-Mimetic Treatment for Modulation of Spasticity

receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments.Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2–3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene.These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel and highly effective anti-spasticity treatment which is associated with minimal side effects and is restricted to GAD65-gene over-expressing spinal segments

Mutations in topoisomerase IIβ result in a B cell immunodeficiency.

B cell development is a highly regulated process involving multiple differentiation steps, yet many details regarding this pathway remain unknown. Sequencing of patients with B cell-restricted immunodeficiency reveals autosomal dominant mutations in TOP2B. TOP2B encodes a type II topoisomerase, an essential gene required to alleviate topological stress during DNA replication and gene transcription, with no previously known role in B cell development. We use Saccharomyces cerevisiae, and knockin and knockout murine models, to demonstrate that patient mutations in TOP2B have a dominant negative effect on enzyme function, resulting in defective proliferation, survival of B-2 cells, causing a block in B cell development, and impair humoral function in response to immunization

Ablation of NG2 Proteoglycan Leads to Deficits in Brown Fat Function and to Adult Onset Obesity

Obesity is a major health problem worldwide. We are studying the causes and effects of obesity in C57Bl/6 mice following genetic ablation of NG2, a chondroitin sulfate proteoglycan widely expressed in progenitor cells and also in adipocytes. Although global NG2 ablation delays early postnatal adipogenesis in mouse skin, adult NG2 null mice are paradoxically heavier than wild-type mice, exhibiting larger white fat deposits. This adult onset obesity is not due to NG2-dependent effects on CNS function, since specific ablation of NG2 in oligodendrocyte progenitors yields the opposite phenotype; i.e. abnormally lean mice. Metabolic analysis reveals that, while activity and food intake are unchanged in global NG2 null mice, O2 consumption and CO2 production are decreased, suggesting a decrease in energy expenditure. Since brown fat plays important roles in regulating energy expenditure, we have investigated brown fat function via cold challenge and high fat diet feeding, both of which induce the adaptive thermogenesis that normally occurs in brown fat. In both tests, body temperatures in NG2 null mice are reduced compared to wild-type mice, indicating a deficit in brown fat function in the absence of NG2. In addition, adipogenesis in NG2 null brown pre-adipocytes is dramatically impaired compared to wild-type counterparts. Moreover, mRNA levels for PR domain containing 16 (PRDM16) and peroxisome proliferator-activated receptor γ coactivator (PGC)1-α, proteins important for brown adipocyte differentiation, are decreased in NG2 null brown fat deposits in vivo and NG2 null brown pre-adipocytes in vitro. Altogether, these results indicate that brown fat dysfunction in NG2 null mice results from deficits in the recruitment and/or development of brown pre-adipocytes. As a consequence, obesity in NG2 null mice may occur due to disruptions in brown fat-dependent energy homeostasis, with resulting effects on lipid storage in white adipocytes

Distinct NG2 proteoglycan-dependent roles of resident microglia and bone marrow-derived macrophages during myelin damage and repair.

We used a bone marrow transplantation approach to distinguish the activities of bone marrow-derived macrophages from the activities of central nervous system-resident microglia in phenomena associated with axon demyelination and remyelination. We transplanted wild type or germline NG2 null beta-actin-EGFP expressing bone marrow into irradiated wild type or NG2 null recipient mice, followed by analysis of lysolecithin-induced spinal cord demyelination and remyelination and quantification of Iba-1+/ F4/80+/ EGFP+ macrophages and Iba-1+/ F4/80+/ EGFP- microglia. One week after microinjection of 1% lysolecithin into the spinal cord, wild type recipients receiving NG2 null bone marrow exhibit greatly reduced infiltration of macrophages into lesions, compared to wild type recipients receiving wild type bone marrow. Wild type bone marrow recipients also exhibit larger numbers of demyelinated axons than NG2 null recipients, indicative of macrophage participation in the initial myelin damage. However, wild type bone marrow recipients also exhibit superior myelin repair at 6 weeks post-injury, compared to NG2 null bone marrow recipients, demonstrating the additional importance of macrophages in remyelination. Incompletely repaired lesions in NG2 null bone marrow recipients at 6 weeks post-injury retain elevated numbers of macrophages, in contrast to lower numbers of macrophages in more completely repaired lesions in wild type bone marrow recipients. This suggests that NG2 expression renders macrophages more effective in myelin repair and less likely to promote chronic inflammation. Effective macrophage involvement in myelin repair is due in part to effects on the proliferation and/or recruitment of oligodendrocyte progenitor cells. Reduced numbers of oligodendrocyte progenitors are seen in lesions in NG2 null bone marrow recipients, likely due to deficits in macrophage production of oligodendrocyte progenitor-relevant mitogens and in phagocytosis of inhibitory myelin debris. Microglia also appear to be important for clearance of myelin debris, as indicated by reduced phagocytosis in NG2 null recipients receiving wild type bone marrow

Dissecting the multifactorial nature of demyelinating disease

Chondroitin sulfate proteoglycan-4 (CSPG4) is a surface component of two key cell types (oligodendrocyte progenitor cells (OPCs) and myeloid cells) present in lysolecithin-induced lesions in mouse spinal cord. Two types of CSPG4 manipulations have been used to study the roles of these cells in myelin damage and repair: (1) OPC and myeloid-specific ablation of CSPG4, and (2) transplantation of enhanced green fluorescent protein (EGFP)-labeled progenitors to distinguish between bone marrow-derived macrophages and resident microglia. Ablation of CSPG4 in OPCs does not affect myelin damage, but decreases myelin repair, due to reduced proliferation of CSPG4-null OPCs that diminishes generation of mature oligodendrocytes for remyelination. Ablation of CSPG4 in myeloid cells greatly decreases recruitment of macrophages to spinal cord lesions, resulting in smaller initial lesions, but also in significantly diminished myelin repair. In the absence of macrophage recruitment, OPC proliferation is greatly impaired, again leading to decreased generation of myelinating oligodendrocytes. Macrophages may promote OPC proliferation via phagocytosis of myelin debris and/or secretion of factors that stimulate OPC mitosis. Microglia are not able to substitute for macrophages in promoting OPC proliferation. An additional feature of lesions in myeloid-specific CSPG4 null mice is the persistence of poorly-differentiated platelet-derived growth factor receptor α (PDGFRα) + macrophages that may prolong damage

Persistence of atypical NG2 null macrophages in lesions in WT-KOBM mice.

<p>Sections from lesions at 6-weeks after lysolecithin injection were evaluated for myeloid cell abundance by use of the EGFP marker (green) and immunolabeling for Iba-1 (blue). <b>A-C</b>. Immunolabeling for MBP (red) allows visualization of the extent of myelin repair. A. WT-WTBM. B. KO-WTBM. C. WT-KOBM. IBa-1-positive, EGFP-positive macrophages remain prominent in areas of the WT-KOBM spinal cord in which myelin repair is incomplete. <b>D-F</b>. Immunolabeling for Iba-1 and PDGFRβ (red) in conjunction with the EGFP marker identifies atypical macrophages that express PDGFRβ. D. WT-WTBM. E. KO-WTBM. F. WT-KOBM. Many persistent macrophages in lesions in WT-KOBM mice co-express Iba-1, EGFP, and PDGFRβ (arrows). Scale bar = 100 μm (A-C) and 25 μm (D-F).</p

Phagocytosis of myelin debris by macrophages and microglia.

<p>Sections from spinal cord lesions one week after lysolecithin microinjection were used to evaluate macrophage and microglial phagocytosis of myelin debris according to the strategy described in Materials and Methods. Cells are examined as being positive for (A) phagocytosed MBP (red), (B) EGFP (green), and (C) Iba-1 (blue). The inset area in panels A-C is shown as a computer-edited merged image at higher magnification in panel D. Phagocytic cells positive for both EGFP and Iba1 are identified as bone marrow-derived macrophages (arrow), while phagocytic cells positive for Iba-1 and negative for EGFP are identified as microglia (arrowhead). In panel D, MBP is readily seen in the Iba1-positive microglial cell (arrowhead). MBP is much less apparent in the Iba1/EGFP-positive macrophage (arrow) due to the strong overlap between EGFP and Iba1 that is seen as white in panel D. Nevertheless, the presence of MBP in both the microglial cell and the macrophage can be seen in panel A, and the Image Pro Plus program allows for MBP quantification in both cell types using merged images (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187530#pone.0187530.t002" target="_blank">Table 2</a>). Scale bar = 60 μm in A and 30 μm in B.</p

NG2 expression 1 and 6 weeks after demyelination.

<p>Image Pro Plus 5.1 software was used to quantify NG2 pixels associated with three classes of cells: macrophages, microglial cells, and non-myeloid cells (this includes both OPCs and pericytes). These cell types were identified by immunolabeling according to the following strategy. 1-week after demyelination in wild type (WT) recipient mice that received WT bone marrow (WT-WTBM), the majority of NG2 expression (blue) occurs in non-myeloid OPCs and pericytes (double arrowheads). NG2 (blue) is also expressed by some IBA1 (red) positive, EGFP-positive macrophages (green; arrow) and by some IBA1 positive, EGFP-negative microglia (arrowhead). (B) In contrast, NG2 expression is greatly reduced in lesion sites in NG2 null recipients that received wild type bone marrow (KO-WTBM). (C) In wild type recipients that received NG2 null bone marrow (WT-KOBM), NG2 is seen in non-myeloid cells (double arrowheads) and in microglia (arrowhead), but not in EGFP-positive macrophages. (D) 6-weeks after demyelination in WT-WTBM mice, overall NG2 expression is only about 30% of that seen at 1-week (G) and is seen mainly in OPCs (double arrowhead). NG2 is occasionally seen in IBA1-positive microglia (arrowhead). (E) In KO-WTBM mice at 6-weeks, NG2 is almost completely absent. (F) In WT-KOBM mice at 6-weeks, the small residual NG2 expression is by non-myeloid cells (double arrowheads). NG2 quantification is summarized in panel G, where total NG2 pixel density in WT-WTBM at 1 week after demyelination is defined as 100%. All other values are compared to this standard. Scale bar indicates 30 μm (A-F). * p < 0.05; *** p < 0.001.</p

Chimeric mice from bone marrow transplantation.

<p>Chimeric mice from bone marrow transplantation.</p