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Mesenchymal stem cells (MSCs) have been shown to improve outcomes after neonatal hypoxic-ischemic (HI) brain injury possibly by secretion of growth factors stimulating repair processes. We investigated whether MSCs, modified to secrete specific growth factors, can further enhance recovery. Using an in vitro assay, we show that MSC-secreting brain derived neurotrophic factor (BDNF), epidermal growth factor-like 7 (EGFL7), persephin (PSP), or sonic hedgehog (SHH) regulate proliferation and differentiation of neural stem cells. Moreover, mice that received an intranasal application of 100,000 MSC-BDNF showed significantly improved outcomes as demonstrated by improved motor function and decreased lesion volume compared with mice treated with empty vector (EV) MSCs. Treatment with MSC-EGFL7 improved motor function but had no effect on lesion size. Treatment with MSC-PSP or MSC-SHH neither improved outcome nor reduced lesion size in comparison with MSC-EV-treated mice. Moreover, mice treated with MSC-SHH showed even decreased functional outcomes when compared with those treated with MSC-EV. Treatment with MSC-BDNF induced cell proliferation in the ischemic hemisphere lasting at least 18 days after MSC administration, whereas treatment with MSC-EV did not. These data suggest that gene-modified cell therapy might be a useful approach to consider for treatment of neonatal HI brain damage. However, care must be taken when selecting the agent to overexpress.

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates cellular signaling via desensitization of GPCRs and by direct interaction with intracellular signaling molecules. We recently described that ischemic brain injury decreases cerebral GRK2 levels. Here we studied the effect of astrocyte GRK2-deficiency on neonatal brain damage in vivo. As astrocytes protect neurons by taking up glutamate via plasma-membrane transporters, we also studied the effect of GRK2 on the localization of the GLutamate ASpartate Transporter (GLAST). Brain damage induced by hypoxia-ischemia was significantly reduced in GFAP-GRK2(+/-) mice, which have a 60% reduction in astrocyte GRK2 compared to GFAP-WT littermates. In addition, GRK2-deficient astrocytes have higher plasma-membrane levels of GLAST and an increased capacity to take up glutamate in vitro. In search for the mechanism by which GRK2 regulates GLAST expression, we observed increased GFAP levels in GRK2-deficient astrocytes. GFAP and the cytoskeletal protein ezrin are known regulators of GLAST localization. In line with this evidence, GRK2-deficiency reduced phosphorylation of the GRK2 substrate ezrin and enforced plasma-membrane GLAST association after stimulation with the group I mGluR-agonist DHPG. When ezrin was silenced, the enhanced plasma-membrane GLAST association in DHPG-exposed GRK2-deficient astrocytes was prevented. In conclusion, we identified a novel role of astrocyte GRK2 in regulating plasma-membrane GLAST localization via an ezrin-dependent route. We demonstrate that the 60% reduction in astrocyte GRK2 protein level that is observed in GFAP-GRK2(+/-) mice is sufficient to significantly reduce neonatal ischemic brain damage. These findings underline the critical role of GRK2 regulation in astrocytes for dampening the extent of brain damage after ischemia., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The ubiquitously expressed kinase GRK2 protects against cellular overstimulation by desensitizing G protein-coupled receptors and regulating intracellular signaling. Recently, we described that hypoxia-ischemia (HI)-induced brain damage was accelerated and increased in GRK2(+/-) neonatal mice. Using Cre-Lox technology we now investigated the role of decreased GRK2 in only microglia/macrophages or forebrain neurons in development of HI brain injury. Low GRK2 in microglia/macrophages (LysM-GRK2(f/+) mice) was sufficient to accelerate onset of HI damage, without affecting the severity of brain injury at 24h post-HI as compared to LysM-GRK2(+/+) littermates. Consistently, the ipsilateral hemisphere of GRK2(+/-) mice contained microglia with a more rounded phenotype compared to WT mice at 3h post-HI. Inhibition of microglial/macrophage activity by minocycline treatment prevented the early onset of HI injury in GRK2(+/-) mice. In vitro, primary GRK2(+/-) microglia stimulated with LPS produced more TNF-alpha than WT microglia via a p38-dependent pathway. In vivo, HI-induced cerebral p38 activation and TNF-alpha production were increased in GRK2(+/-) mice or in LysM-GRK2(f/+) mice. Our findings indicate that low GRK2 in microglia/macrophages accelerates brain damage via a GRK2/p38/TNF-alpha-dependent pathway. Reduced GRK2 only in forebrain neurons (CamKIIalpha-GRK2(f/+) mice) significantly increased severity of HI brain damage without affecting the onset of brain damage. In conclusion, our data indicate that low GRK2 in microglia/macrophages facilitates activation of these cells which may contribute to the earlier onset of cerebral HI injury associated with increased p38 phosphorylation and TNF-alpha production. The level of GRK2 in neurons is crucial for determining the ultimate severity of HI damage in the newborn brain., (2009 Elsevier Inc. All rights reserved.)