Investigating Bacterial Sources of Toxicity as an Environmental Contributor to Dopaminergic Neurodegeneration

Parkinson disease (PD) involves progressive neurodegeneration, including loss of dopamine (DA) neurons from the substantia nigra. Select genes associated with rare familial forms of PD function in cellular pathways, such as the ubiquitin-proteasome system (UPS), involved in protein degradation. The misfolding and accumulation of proteins, such as α-synuclein, into inclusions termed Lewy Bodies represents a clinical hallmark of PD. Given the predominance of sporadic PD among patient populations, environmental toxins may induce the disease, although their nature is largely unknown. Thus, an unmet challenge surrounds the discovery of causal or contributory neurotoxic factors that could account for the prevalence of sporadic PD. Bacteria within the order Actinomycetales are renowned for their robust production of secondary metabolites and might represent unidentified sources of environmental exposures. Among these, the aerobic genera, Streptomyces, produce natural proteasome inhibitors that block protein degradation and may potentially damage DA neurons. Here we demonstrate that a metabolite produced by a common soil bacterium, S. venezuelae, caused DA neurodegeneration in the nematode, Caenorhabditis elegans, which increased as animals aged. This metabolite, which disrupts UPS function, caused gradual degeneration of all neuronal classes examined, however DA neurons were particularly vulnerable to exposure. The presence of DA exacerbated toxicity because neurodegeneration was attenuated in mutant nematodes depleted for tyrosine hydroxylase (TH), the rate-limiting enzyme in DA production. Strikingly, this factor caused dose-dependent death of human SH-SY5Y neuroblastoma cells, a dopaminergic line. Efforts to purify the toxic activity revealed that it is a highly stable, lipophilic, and chemically unique small molecule. Evidence of a robust neurotoxic factor that selectively impacts neuronal survival in a progressive yet moderate manner is consistent with the etiology of age-associated neurodegenerative diseases. Collectively, these data suggest the potential for exposures to the metabolites of specific common soil bacteria to possibly represent a contributory environmental component to PD

Neurodegeneration occurs in <i>C. elegans</i> following exposure to the proteasome inhibitor MG-132.

<p>An isogenic worm strain expressing GFP in DA neurons was examined for DA neurodegeneration every two days. Significant DA neurodegeneration occurred after eight days of continuous exposure to MG-132 in comparison to the solvent control (*<i>P</i><0.05; ANOVA). All data in this figure are represented as mean worms with neurodegeneration +/− S.E.M.</p

Neurodegeneration occurs in <i>C. elegans</i> following exposure to <i>S. venezuelae</i> conditioned medium.

<p>A. An isogenic worm strain expressing GFP in DA neurons was examined for DA neurodegeneration four and six days after exposure to <i>Streptomyces</i> spp. conditioned media. Significant DA neurodegeneration only occurred from exposure to <i>S. venezuelae</i> conditioned medium (*<i>P</i><0.05; ANOVA). B. Photomicrographs of GFP-labeled DA neurons from <i>C. elegans</i> exposed to bacterial conditioned medium for six days. All six anterior DA neurons in <i>C. elegans</i> exhibit degenerative changes following exposure to <i>S. venezuelae</i> but not <i>E. coli</i> (control) medium (the four CEP class of DA neurons and the two ADE class of DA neurons are indicated with arrows and arrowheads, respectively). C. Populations of isogenic worm strains expressing GFP exclusively in 5-HT, GABA, ACh, Glut, and DA (+ and –TH) neuronal classes were examined for neurodegeneration. The only animals that displayed significant neurodegeneration after six days exposure to <i>S. venezuelae</i> conditioned medium were those in which the DA (+TH) neurons were analyzed (*<i>P</i><0.01; ANOVA). These data were standardized against the amount of degeneration observed from exposure to <i>E. coli</i> control conditioned medium. D. At eight days of exposure to <i>S. venezuelae</i> conditioned medium, all neuronal classes examined exhibited significant degeneration (*<i>P</i><0.01; ANOVA). These data were standardized against the amount of degeneration observed from exposure to <i>E. coli</i> control conditioned medium. Because each neuronal class contains varying numbers of neurons, this analysis was based on the percentage of degenerating neurons (not degenerating worms, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007227#pone-0007227-g001" target="_blank">Fig. 1C</a>) to compensate for differences in total neuron numbers. Furthermore, DA neurons still exhibited significantly more degeneration than other neuronal classes (*<i>P</i><0.05; ANOVA). All graphical data in this figure are represented as mean degeneration +/− S.E.M. Scale bar = 50 µM.</p

<i>C. elegans</i> neurons cultured <i>in vitro</i> display enhanced levels of degeneration in response to exposure to <i>S. venezuelae</i> conditioned medium.

<p>A. GABA neurons degenerate slowly in culture when compared to DA neurons. B. DA neurons degenerate rapidly in culture. Almost all wild-type DA neurons (+TH) are degenerated after five days of continuous exposure to the conditioned medium. A substantial proportion of DA neurons from <i>cat-2</i> mutant worms (–TH) degenerate as well. C. Photomicrographs that capture the same DA neurons (+ and –TH) over the course of several days exposure to the <i>S. venezuelae</i> factor. There is one less image for DA+ neurons because this neuron had degenerated by day four and was not visible. Arrows depict degeneration in cell processes. All graphical data in this figure are represented as mean degeneration relative to <i>E. coli</i> conditioned medium controls (control degeneration was always <2%). Scale bar = 5 µM.</p

The <i>S. venezuelae</i> factor does not enhance expression of <i>hsp-16</i>, a small heat shock protein.

<p>LacZ expression is driven from the <i>hsp-16</i> promoter in an isogenic worm strain. X-gal staining was used to examine expression of β-galactosidase (β-gal) in these animals. A. <i>hsp-16</i> expression occurs in response to specific stressors, such as CdCl₂. In contrast, <i>hsp-16</i> expression is minimal within populations of <i>C. elegans</i> exposed to <i>S. venezuelae</i> or <i>E. coli</i> conditioned media for six days (*<i>P</i><0.001; ANOVA). These data are represented as mean worms positively stained with X-gal +/− S.E.M. B. Worms exposed to the proteasome inhibitor MG-132 for 6 days display minimal expression of <i>hsp-16</i>, in contrast to the CdCl₂ control (*<i>P</i><0.001; Fisher Exact Test). C. Photomicrographs depicting representative worms exposed to <i>E. coli</i> or <i>S. venezuelae</i> conditioned medium, MG-132, or CdCl_2.<i>C. elegans</i> exposed to CdCl₂ exhibited prominent hypodermal induction of β-gal (arrow). Scale bar = 100 µM.</p

Human SH-SY5Y neuroblastoma cells exhibit cell death following exposure to <i>S. venezuelae</i> conditioned medium.

<p>Cell death was measured using LDH release; it was significantly enhanced when cells were exposed to <i>S. venezuelae</i> in comparison to <i>S. coelicolor</i>. This experiment was performed three times, in duplicate (n = 6) (*<i>P</i><0.001; ANOVA). These data depict one independent experiment that is representative of the others, whereby mean LDH release is displayed as +/− S. D.</p

The <i>S. venezuelae</i> neurodegenerative factor is lipophilic and stable.

<p>A. Boiling conditioned media for 30 minutes or digestion with proteinase K had no discernable effect on the neurodegenerative effect of the <i>S. venezuelae</i> factor. All data in this figure are represented as percentage of mean worms exhibiting DA neurodegeneration. B. Partitioning with ethyl acetate (EtoAc) resulted in activity separation within the organic but not the aqueous phase (compare ethyl acetate solvent only with aqueous portion). Further partitioning with dichloromethane (DcM) also resulted in the activity separating in the lipophilic portion. In all cases, extracts were dried and resolubilized with ethyl acetate (solvent only control), as it is non-lethal to <i>C. elegans</i>. The amount of DA neurodegeneration observed in the conditioned media control was lower than previously reported because 34% less factor was used in each experiment (to ensure final ethyl acetate concentrations were below 1.5%).</p

Ubiquitin-related degradation is impaired in <i>C. elegans</i> DA neurons following exposure to the <i>S. venezuelae</i> factor.

<p>A. Isogenic worms expressing a CFP::CL-1 degron fusion protein within DA neurons were exposed to the proteasome inhibitor MG-132. Mean pixel intensity of CFP fluorescence is significantly higher when the proteasome in inactived (*<i>P</i><0.05; Fisher Exact Test). B. CFP fluorescence is also significantly higher following exposure to <i>S. venezuelae</i> conditioned medium when compared to <i>E. coli</i> medium (*<i>P</i><0.05; Fisher Exact Test). All data in this figure are represented as mean pixel intensities [in arbitrary units (a.u.)] +/− S.E.M.</p