Erythropoietin and small molecule agonists of the tissue-protective erythropoietin receptor increase FXN expression in neuronal cells in vitro and in Fxn-deficient KIKO mice in vivo.

Friedreich's ataxia (FA) is a progressive neurodegenerative disease caused by reduced levels of the mitochondrial protein frataxin (FXN). Recombinant human erythropoietin (rhEPO) increased FXN protein in vitro and in early clinical studies, while no published reports evaluate rhEPO in animal models of FA. STS-E412 and STS-E424 are novel small molecule agonists of the tissue-protective, but not the erythropoietic EPO receptor. We find that rhEPO, STS-E412 and STS-E424 increase FXN expression in vitro and in vivo. RhEPO, STS-E412 and STS-E424 increase FXN by up to 2-fold in primary human cortical cells and in retinoic-acid differentiated murine P19 cells. In primary human cortical cells, the increase in FXN protein was accompanied by an increase in FXN mRNA, detectable within 4 h. RhEPO and low nanomolar concentrations of STS-E412 and STS-E424 also increase FXN in normal and FA patient-derived PBMC by 20%-40% within 24 h, an effect that was comparable to that by HDAC inhibitor 4b. In vivo, STS-E412 increased Fxn mRNA and protein in wild-type C57BL6/j mice. RhEPO, STS-E412, and STS-E424 increase FXN expression in the heart of FXN-deficient KIKO mice. In contrast, FXN expression in the brains of KIKO mice increased following treatment with STS-E412 and STS-E424, but not following treatment with rhEPO. Unexpectedly, rhEPO-treated KIKO mice developed severe splenomegaly, while no splenomegaly was observed in STS-E412- or STS-E424-treated mice. RhEPO, STS-E412 and STS-E424 upregulate FXN expression in vitro at equal efficacy, however, the effects of the small molecules on FXN expression in the CNS are superior to rhEPO in vivo.

Discovery and Characterization of Nonpeptidyl Agonists of the Tissue-Protective Erythropoietin Receptor.

Erythropoietin (EPO) and its receptor are expressed in a wide variety of tissues, including the central nervous system. Local expression of both EPO and its receptor is upregulated upon injury or stress and plays a role in tissue homeostasis and cytoprotection. High-dose systemic administration or local injection of recombinant human EPO has demonstrated encouraging results in several models of tissue protection and organ injury, while poor tissue availability of the protein limits its efficacy. Here, we describe the discovery and characterization of the nonpeptidyl compound STS-E412 (2-[2-(4-chlorophenoxy)ethoxy]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine), which selectively activates the tissue-protective EPO receptor, comprising an EPO receptor subunit (EPOR) and the common ß-chain (CD131). STS-E412 triggered EPO receptor phosphorylation in human neuronal cells. STS-E412 also increased phosphorylation of EPOR, CD131, and the EPO-associated signaling molecules JAK2 and AKT in HEK293 transfectants expressing EPOR and CD131. At low nanomolar concentrations, STS-E412 provided EPO-like cytoprotective effects in primary neuronal cells and renal proximal tubular epithelial cells. The receptor selectivity of STS-E412 was confirmed by a lack of phosphorylation of the EPOR/EPOR homodimer, lack of activity in off-target selectivity screening, and lack of functional effects in erythroleukemia cell line TF-1 and CD34(+) progenitor cells. Permeability through artificial membranes and Caco-2 cell monolayers in vitro and penetrance across the blood-brain barrier in vivo suggest potential for central nervous system availability of the compound. To our knowledge, STS-E412 is the first nonpeptidyl, selective activator of the tissue-protective EPOR/CD131 receptor. Further evaluation of the potential of STS-E412 in central nervous system diseases and organ protection is warranted.

Glycans flanking the hypervariable connecting peptide between the A and B strands of the V1/V2 domain of HIV-1 gp120 confer resistance to antibodies that neutralize CRF01_AE viruses.

Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain ß-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.

Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system.

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of alpha-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to alpha-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates alpha-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited alpha-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in alpha-synuclein phosphorylation in central nervous system.