Bone marrow-derived microglia-based neurturin delivery protects against dopaminergic neurodegeneration in a mouse model of Parkinson's disease.

Although neurotrophic factors have long been recognized as potent agents for protecting against neuronal degeneration, clinical success in treating Parkinson's disease and other neurodegenerative disorders has been hindered by difficulties in delivery of trophic factors across the blood brain barrier (BBB). Bone marrow hematopoietic stem cell-based gene therapy is emerging as a promising tool for overcoming drug delivery problems, as myeloid cells can cross the BBB and are recruited in large numbers to sites of neurodegeneration, where they become activated microglia that can secrete trophic factors. We tested the efficacy of bone marrow-derived microglial delivery of neurturin (NTN) in protecting dopaminergic neurons against neurotoxin-induced death in mice. Bone marrow cells were transduced ex vivo with lentivirus expressing the NTN gene driven by a synthetic macrophage-specific promoter. Infected bone marrow cells were then collected and transplanted into recipient animals. Eight weeks after transplantation, the mice were injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropuridine (MPTP) for seven days to induce dopaminergic neurodegeneration. Microglia-mediated NTN delivery dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and their terminals in the striatum. Microglia-mediated NTN delivery also induced significant recovery of synaptic marker staining in the striatum of MPTP-treated animals. Functionally, NTN treatment restored MPTP-induced decline in general activity, rearing behavior, and food intake. Thus, bone marrow-derived microglia can serve as cellular vehicles for sustained delivery of neurotrophic factors capable of mitigating dopaminergic injury.

Dipeptidylpeptidase 4 negatively regulates colony-stimulating factor activity and stress hematopoiesis.

Enhancement of hematopoietic recovery after radiation, chemotherapy, or hematopoietic stem cell (HSC) transplantation is clinically relevant. Dipeptidylpeptidase (DPP4) cleaves a wide variety of substrates, including the chemokine stromal cell-derived factor-1 (SDF-1). In the course of experiments showing that inhibition of DPP4 enhances SDF-1-mediated progenitor cell survival, ex vivo cytokine expansion and replating frequency, we unexpectedly found that DPP4 has a more general role in regulating colony-stimulating factor (CSF) activity. DPP4 cleaved within the N-termini of the CSFs granulocyte-macrophage (GM)-CSF, G-CSF, interleukin-3 (IL-3) and erythropoietin and decreased their activity. Dpp4 knockout or DPP4 inhibition enhanced CSF activities both in vitro and in vivo. The reduced activity of DPP4-truncated versus full-length human GM-CSF was mechanistically linked to effects on receptor-binding affinity, induction of GM-CSF receptor oligomerization and signaling capacity. Hematopoiesis in mice after radiation or chemotherapy was enhanced in Dpp4(-/-) mice or mice receiving an orally active DPP4 inhibitor. DPP4 inhibition enhanced engraftment in mice without compromising HSC function, suggesting the potential clinical utility of this approach.

STAT3-dependent IL-21 production from T helper cells regulates hematopoietic progenitor cell homeostasis.

The contribution of specific cell types to the production of cytokines that regulate hematopoiesis is still not well defined. We have previously identified T cell-dependent regulation of hematopoietic progenitor cell (HPC) numbers and cycling. In this report, we demonstrated that HPC activity is decreased in mice with STAT3-deficient T cells, a phenotype that is not because of decreased expression of IL-17 or RORγt. STAT3 expression in T cells was required for IL-21 production by multiple T helper subsets, and neutralization of IL-21 resulted in decreased HPC activity identical to that in mice with STAT3-deficient T cells. Importantly, injection of IL-21 rescued HPC activity in mice with STAT3-deficient T cells. Thus, STAT3-dependent IL-21 production in T cells is required for HPC homeostasis.

Ex vivo rapamycin treatment of human cord blood CD34+ cells enhances their engraftment of NSG mice.

Since cord blood (CB) has become a commonly used source of transplantable hematopoietic stem (HSC) and hematopoietic progenitor cells (HPC), there has been a need to overcome the limited HSC and HPC numbers available to transplant from a single CB, especially for adult recipients. Our laboratory previously demonstrated that Rheb2 overexpression significantly impaired the repopulating ability of HSC. Since overexpression of Rheb2 leads to increased signaling through mTOR, we examined the effect of the mTOR inhibitor rapamycin ex vivo on cytokine expanded CD34(+) CB cells for the engraftment of these cells in non-obese diabetic, severe combined immunodeficient, IL-2 receptor γ chain null (NSG) mice. We observed significant enhancement in engraftment of the CB treated ex vivo with cytokines in the presence of rapamycin prior to transplant, effects seen in primary as well as secondary transplants. These pre-clinical results suggest a positive role for rapamycin during ex vivo culture of CB SCID repopulating cells/HSC.

Mad2 haploinsufficiency protects hematopoietic progenitor cells subjected to cell-cycle stress in vivo and to inhibition of redox function of Ape1/Ref-1 in vitro.

OBJECTIVE: Cell-cycle checkpoints guarantee movement through the cell cycle. Mitotic arrest deficiency 2 (Mad2), a mitotic checkpoint protein, appears crucial for generating the wait anaphase signal to prevent onset of anaphase. We evaluated effects of Mad2 haploinsufficiency on hematopoietic stem (HSC) and progenitor (HPC) function in response to stress. MATERIALS AND METHODS: We studied effects of Mad2(+/-) on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2(+/-) mice. RESULTS: Mad2(+/-) HPCs were protected from cytotoxic effects in vivo of a cell-cycle-specific agent, Ara-C, events consistent with Mad2(+/-) HPCs being in a slow or noncycling state, but not from recovery of functional HPC after treatment with non-cycle-specific cyclophosphamide or sublethal irradiation. There were no differences in phenotyped HSCs in Mad2(+/-) &Mad2(+/+) mice, information confirmed by no changes in short- or long-term repopulating HSC assay. To better understand Mad2(+/-) HPC function, E3330, a cytostatic agent, was used to assess redox function of Ape1/Ref-1; colony growth was examined under 5% and 20% O(2) tension. Mad2(+/-) HPCs were less responsive to E3330 than Mad2(+/+) HPCs, and E3330 was more effective under lowered O(2) tension. Mad2(+/-) HPCs were not enhanced at lowered oxygen, as were Mad2(+/+) HPCs. CONCLUSIONS: Our studies have unexpectedly found that Mad2 haploinsufficiency is protective in the presence of a cycle-specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro.