NME1 Protects Against Neurotoxin-, α-Synuclein- and LRRK2-Induced Neurite Degeneration in Cell Models of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disease characterised by the progressive degeneration of midbrain dopaminergic neurons, coupled with the intracellular accumulation of α-synuclein. Axonal degeneration is a central part of the pathology of PD. While the majority of PD cases are sporadic, some are genetic; the G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is the most common genetic form. The application of neurotrophic factors to protect dopaminergic neurons is a proposed experimental therapy. One such neurotrophic factor is growth differentiation factor (GDF)5. GDF5 is a dopaminergic neurotrophic factor that has been shown to upregulate the expression of a protein called nucleoside diphosphate kinase A (NME1). However, whether NME1 is neuroprotective in cell models of axonal degeneration of relevance to PD is unknown. Here we show that treatment with NME1 can promote neurite growth in SH-SY5Y cells, and in cultured dopaminergic neurons treated with the neurotoxin 6-hydroxydopamine (6-OHDA). Similar effects of NME1 were found in SH-SY5Y cells and dopaminergic neurons overexpressing human wild-type α-synuclein, and in stable SH-SY5Y cell lines carrying the G2019S LRRK2 mutation. We found that the effects of NME1 require the RORα/ROR2 receptors. Furthermore, increased NF-κB-dependent transcription was partially required for the neurite growth-promoting effects of NME1. Finally, a combined bioinformatics and biochemical analysis of the mitochondrial oxygen consumption rate revealed that NME1 enhanced mitochondrial function, which is known to be impaired in PD. These data show that recombinant NME1 is worthy of further study as a potential therapeutic agent for axonal protection in PD.

The Potential Role of Human NME1 in Neuronal Differentiation of Porcine Mesenchymal Stem Cells: Application of NB-hNME1 as a Human NME1 Suppressor.

This study aimed to investigate the effects of the human macrophage (MP) secretome in cellular xenograft rejection. The role of human nucleoside diphosphate kinase A (hNME1), from the secretome of MPs involved in the neuronal differentiation of miniature pig adipose tissue-derived mesenchymal stem cells (mp AD-MSCs), was evaluated by proteomic analysis. Herein, we first demonstrate that hNME1 strongly binds to porcine ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 (pST8SIA1), which is a ganglioside GD3 synthase. When hNME1 binds with pST8SIA1, it induces degradation of pST8SIA1 in mp AD-MSCs, thereby inhibiting the expression of ganglioside GD3 followed by decreased neuronal differentiation of mp AD-MSCs. Therefore, we produced nanobodies (NBs) named NB-hNME1 that bind to hNME1 specifically, and the inhibitory effect of NB-hNME1 was evaluated for blocking the binding between hNME1 and pST8SIA1. Consequently, NB-hNME1 effectively blocked the binding of hNME1 to pST8SIA1, thereby recovering the expression of ganglioside GD3 and neuronal differentiation of mp AD-MSCs. Our findings suggest that mp AD-MSCs could be a potential candidate for use as an additive, such as an immunosuppressant, in stem cell transplantation.

Characterization of Functional Domains in NME1L Regulation of NF-κB Signaling.

NME1 is a well-known metastasis suppressor which has been reported to be downregulated in some highly aggressive cancer cells. Although most studies have focused on NME1, the NME1 gene also encodes the protein (NME1L) containing N-terminal 25 extra amino acids by alternative splicing. According to previous studies, NME1L has potent anti-metastatic activity, in comparison with NME1, by interacting with IKKß and regulating its activity. In the present study, we tried to define the role of the N-terminal 25 amino acids of NME1L in NF-κB activation signaling. Unfortunately, the sequence itself did not interact with IKKß, suggesting that it may be not enough to constitute the functional structure. Further construction of NME1L fragments and biochemical analysis revealed that N-terminal 84 residues constitute minimal structure for homodimerization, IKKß interaction and regulation of NF-κB signaling. The inhibitory effect of the fragment on cancer cell migration and NF-κB-stimulated gene expression was equivalent to that of whole NME1L. The data suggest that the N-terminal 84 residues may be a core region for the anti-metastatic activity of NME1L. Based on this result, further structural analysis of the binding between NME1L and IKKß may help in understanding the anti-metastatic activity of NME1L and provide direction to NME1L and IKKß-related anti-cancer drug design.

MUC1* ligand, NM23-H1, is a novel growth factor that maintains human stem cells in a more naïve state.

We report that a single growth factor, NM23-H1, enables serial passaging of both human ES and iPS cells in the absence of feeder cells, their conditioned media or bFGF in a fully defined xeno-free media on a novel defined, xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more "naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1, we override the stem cell's inherent programming that turns off pluripotency and trick the cells into continuously replicating as pluripotent stem cells. Dimeric NM23-H1 binds to and dimerizes the extra cellular domain of the MUC1* transmembrane receptor which stimulates growth and promotes pluripotency. Inhibition of the NM23-H1/MUC1* interaction accelerates differentiation and causes a spike in miR-145 expression which signals a cell's exit from pluripotency.

Cell-permeable NM23 blocks the maintenance and progression of established pulmonary metastasis.

Occult metastases are a major cause of cancer mortality, even among patients undergoing curative resection. Therefore, practical strategies to target the growth and persistence of already established metastases would provide an important advance in cancer treatment. Here, we assessed the potential of protein therapy using a cell permeable NM23-H1 metastasis suppressor protein. Hydrophobic transduction domains developed from a screen of 1,500 signaling peptide sequences enhanced the uptake of the NM23 protein by cultured cells and systemic delivery to animal tissues. The cell-permeable (CP)-NM23 inhibited metastasis-associated phenotypes in tumor cell lines, blocked the establishment of lung metastases, and cleared already established pulmonary metastases, significantly prolonging the survival of tumor-bearing animals. Therefore, these results establish the potential use of cell-permeable metastasis suppressors as adjuvant therapy against disseminated cancers.

Nm23-h1 indirectly promotes the survival of acute myeloid leukemia blast cells by binding to more mature components of the leukemic clone.

Nm23-H1 plays complex roles in the development of diverse cancers including breast carcinoma, high-grade lymphomas, and acute myeloid leukemia (AML). In the case of AML and lymphomas, serum Nm23-H1 protein is elevated with the highest levels correlating with poorest prognosis. A recent study identified that this association is most likely causal in AML and that Nm23-H1 acts as an AML cell survival factor. In this study, we report heterogeneity in the ability of AML samples to bind and respond to Nm23-H1, and we offer evidence that binding is essential for improved survival. Further, we show that the subset of AMLs that bind Nm23-H1 do not do so through the putative Nm23-H1 receptor MUC1*. Although rNm23-H1 promoted the survival of the most primitive blasts within responding AMLs, it was not these cells that actually bound the protein. Instead, rNm23-H1 bound to more mature CD34(lo)/CD34(-) and CD11b(+) cells, revealing an indirect survival benefit of Nm23-H1 on primitive blasts. In support of this finding, the survival of purified blast cells was enhanced by medium conditioned by more mature cells from the clone that had been stimulated by rNm23-H1. Levels of interleukin 1ß (IL1ß) and IL6 in rNm23-H1 conditioned medium mirrored the potency of the conditioned media to promote blast cell survival. Furthermore, Nm23-H1 expression was significantly associated with IL1ß and IL6 expression in primary uncultured AML samples. These findings have implications for the role of Nm23-H1 in AML and its use as a prognostic marker. Additionally, they offer the first evidence of novel cross-talk between cell populations within the tumor clone.

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity.

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.

Extracellular NM23-H1 protein inhibits the survival of primary cultured normal human peripheral blood mononuclear cells and activates the cytokine production.

An elevated serum level of NM23-H1 protein is found in acute myelogenous leukemia (AML) and predicts a poor treatment outcome for AML patients. To investigate the potential pathological link between the elevated serum level of this protein and poor prognosis, we examined the extracellular effects of recombinant NM23-H1 protein on the in vitro survival of primary cultured normal peripheral blood mononuclear cells (PBMNC) at concentrations equivalent to the levels found in the serum of AML patients. Extracellular NM23-H1 protein inhibited the in vitro survival of PBMNC and promoted the production of various cytokines, such as GM-CSF and IL-1beta, which in fact promoted the growth of primary cultured AML cells. These findings indicate a novel biological action of extracellular NM23-H1 and its association with poor prognosis of patients with elevated serum levels of NM23-H1 protein. These results suggest an important role of extracellular NM23-H1 in the malignant progression of leukemia and a potential therapeutic target for these malignancies.