A novel iron (II) preferring dopamine agonist chelator D-607 significantly suppresses α-syn- and MPTP-induced toxicities in vivo.

Here, we report the characterization of a novel hybrid D2/D3 agonist and iron (II) specific chelator, D-607, as a multi-target-directed ligand against Parkinson's disease (PD). In our previously published report, we showed that D-607 is a potent agonist of dopamine (DA) D2/D3 receptors, exhibits efficacy in a reserpinized PD animal model and preferentially chelates to iron (II). As further evidence of its potential as a neuroprotective agent in PD, the present study reveals D-607 to be protective in neuronal PC12 cells against 6-OHDA toxicity. In an in vivo Drosophila melanogaster model expressing a disease-causing variant of α-synuclein (α-Syn) protein in fly eyes, the compound was found to significantly suppress toxicity compared to controls, concomitant with reduced levels of aggregated α-Syn. Furthermore, D-607 was able to rescue DAergic neurons from MPTP toxicity in mice, a well-known PD neurotoxicity model, following both sub-chronic and chronic MPTP administration. Mechanistic studies indicated that possible protection of mitochondria, up-regulation of hypoxia-inducible factor, reduction in formation of α-Syn aggregates and antioxidant activity may underlie the observed neuroprotection effects. These observations strongly suggest that D-607 has potential as a promising multifunctional lead molecule for viable symptomatic and disease-modifying therapy for PD.

Interaction of the polyglutamine protein ataxin-3 with Rad23 regulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3.

Polyglutamine (polyQ) repeat expansion in the deubiquitinase ataxin-3 causes neurodegeneration in Spinocerebellar Ataxia Type 3 (SCA3), one of nine inherited, incurable diseases caused by similar mutations. Ataxin-3's degradation is inhibited by its binding to the proteasome shuttle Rad23 through ubiquitin-binding site 2 (UbS2). Disrupting this interaction decreases levels of ataxin-3. Since reducing levels of polyQ proteins can decrease their toxicity, we tested whether genetically modulating the ataxin-3-Rad23 interaction regulates its toxicity in Drosophila. We found that exogenous Rad23 increases the toxicity of pathogenic ataxin-3, coincident with increased levels of the disease protein. Conversely, reducing Rad23 levels alleviates toxicity in this SCA3 model. Unexpectedly, pathogenic ataxin-3 with a mutated Rad23-binding site at UbS2, despite being present at markedly lower levels, proved to be more pathogenic than a disease-causing counterpart with intact UbS2. Additional studies established that the increased toxicity upon mutating UbS2 stems from disrupting the autoprotective role that pathogenic ataxin-3 has against itself, which depends on the co-chaperone, DnaJ-1. Our data reveal a previously unrecognized balance between pathogenic and potentially therapeutic properties of the ataxin-3-Rad23 interaction; they highlight this interaction as critical for the toxicity of the SCA3 protein, and emphasize the importance of considering protein context when pursuing suppressive avenues.

Inhibition of alpha-synuclein aggregation by multifunctional dopamine agonists assessed by a novel in vitro assay and an in vivo Drosophila synucleinopathy model.

Aggregation of alpha synuclein (α-syn) leading to dopaminergic neuronal death has been recognized as one of the main pathogenic factors in the initiation and progression of Parkinson's disease (PD). Consequently, α-syn has been targeted for the development of therapeutics for PD. We have developed a novel assay to screen compounds with α-syn modulating properties by mimicking recent findings from in vivo animal studies involving intrastriatal administration of pre-formed fibrils in mice, resulting in increased α-syn pathology accompanying the formation of Lewy-body (LB) type inclusions. We found that in vitro generated α-syn pre-formed fibrils induce seeding of α-syn monomers to produce aggregates in a dose-and time-dependent manner under static conditions in vitro. These aggregates were toxic towards rat pheochromocytoma cells (PC12). Our novel multifunctional dopamine agonists D-519 and D-520 exhibited significant neuroprotection in this assay, while their parent molecules did not. The neuroprotective properties of our compounds were further evaluated in a Drosophila model of synucleinopathy. Both of our compounds showed protective properties in fly eyes against the toxicity caused by α-syn. Thus, our in vitro results on modulation of aggregation and toxicity of α-syn by our novel assay were further validated with the in vivo experiments.

Effects of low oxygen levels on G2-specific cytogenetic low-dose hyper-radiosensitivity in irradiated human cells.

Low-dose hyper-radiosensitivity (HRS) has been reported in normal human lymphoblastoid cell lines for exposures at ≤ 20 cGy, but the cytogenetic effects of oxygen (O2 ) levels in tissue culture medium on HRS have not been evaluated. We asked whether HRS was lost in G2-irradiated cells grown in atmospheres of 2.5% or 5% O2 , compared to responses by cells cultured in ambient O2 (21%). The results indicate a loss of HRS when cells are cultured and irradiated either in 2.5% or 5% O2 . We then evaluated whether low O2 levels either before or after exposure were responsible for the loss of HRS. For cells irradiated in 5% O2 , subsequent immediate re-oxygenation to ambient O2 levels restored the HRS effect, while cells cultured and irradiated at ambient O2 levels and then transferred to 5% O2 exhibited little or no HRS, indicating that ambient O2 levels after, but not before, radiation substantially affect the amounts of cytogenetic damage. HRS was not observed when cells were irradiated in G1. At doses of 40-400 cGy there was significantly less cytogenetic damage when cells were recovering from radiation at low O2 levels than at ambient O2 levels. Here we provide the first cytogenetic evidence for the loss of HRS at low O2 levels in G2-irradiated cells; these results suggest that at low O2 levels for all doses evaluated there is either less damage to DNA, perhaps because of lower amounts of reactive oxygen species, or that DNA damage repair pathways are activated more efficiently.

Cytogenetic characterization of low-dose hyper-radiosensitivity in Cobalt-60 irradiated human lymphoblastoid cells.

The dose-effect relationships of cells exposed to ionizing radiation are frequently described by linear quadratic (LQ) models over an extended dose range. However, many mammalian cell lines, when acutely irradiated in G2 at doses ≤0.3Gy, show hyper-radiosensitivity (HRS) as measured by reduced clonogenic cell survival, thereby indicating greater cell lethality than is predicted by extrapolation from high-dose responses. We therefore hypothesized that the cytogenetic response in G2 cells to low doses would also be steeper than predicted by LQ extrapolation from high doses. We tested our hypothesis by exposing four normal human lymphoblastoid cell lines to 0-400cGy of Cobalt-60 gamma radiation. The cytokinesis block micronucleus assay was used to determine the frequencies of micronuclei and nucleoplasmic bridges. To characterize the dependence of the cytogenetic damage on dose, univariate and multivariate regression analyses were used to compare the responses in the low- (HRS) and high-dose response regions. Our data indicate that the slope of the response for all four cell lines at ≤20cGy during G2 is greater than predicted by an LQ extrapolation from the high-dose responses for both micronuclei and bridges. These results suggest that the biological consequences of low-dose exposures could be underestimated and may not provide accurate risk assessments following such exposures.