Caenorhabditis elegans as a model system for identifying effectors of α-synuclein misfolding and dopaminergic cell death associated with Parkinson's disease

Protein misfolding and aggregation are key pathological features observed in numerous neurodegenerative diseases, including the misfolding of α-synuclein (α-syn) in Parkinson's disease (PD) and β-amyloid in Alzheimer's disease. While this phenomenon is widely observed, the etiology and progression of these diseases is not fully understood. Furthermore, there is a lack of therapeutic treatments directed at halting the progression and neurodegeneration associated with these diseases. This demands a need for an inexpensive, easy to manipulate multicellular organism to conduct both genetic and chemical screens within to identify factors that may play a pivotal role in the pathology of these diseases. Herein, we describe methodology involved in identifying genetic modifiers of α-syn misfolding and toxicity in the nematode roundworm, Caenorhabditis elegans. Transgenic nematodes engineered to express human α-syn in the body wall muscles or dopaminergic (DA) neurons result in formation of cytoplasmic puncta or DA neurodegeneration, respectively. Using these models, we describe the use of RNA interference (RNAi) and transgenic gene expression to functionally elucidate potential therapeutic gene targets that alter α-syn misfolding and DA neurotoxicity.     

High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction

Background

Therapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death.

Methods

A combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for in vivo treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for in vivo experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation.

Results

No visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain.

Conclusions

H-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.     

Arginase 2 deletion reduces neuro-glial injury and improves retinal function in a model of retinopathy of prematurity

Background

Retinopathy of prematurity (ROP) is a major cause of vision impairment in low birth weight infants. While previous work has focused on defining the mechanisms of vascular injury leading to retinal neovascularization, recent studies show that neurons are also affected. This study was undertaken to determine the role of the mitochondrial arginine/ornithine regulating enzyme arginase 2 (A2) in retinal neuro-glial cell injury in the mouse model of ROP.

Methods and Findings

Studies were performed using wild type (WT) and A2 knockout (A2−/−) mice exposed to Oxygen Induced Retinopathy (OIR). Neuronal injury and apoptosis were assessed using immunohistochemistry, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end) labeling and Western blotting. Electroretinography (ERG) was used to assess retinal function. Neuro-glial injury in WT ROP mice was evident by TUNEL labeling, retinal thinning, decreases in number of rod bipolar cells and glial cell activation as compared with room air controls. Significant reduction in numbers of TUNEL positive cells, inhibition of retinal thinning, preservation of the rod bipolar cells and prevention of glial activation were observed in the A2−/− retinas. Retinal function was markedly impaired in the WT OIR mice as shown by decreases in amplitude of the b-wave of the ERG. This defect was significantly reduced in A2−/− mice. Levels of the pro-apoptotic proteins p53, cleaved caspase 9, cytochrome C and the mitochondrial protein Bim were markedly increased in WT OIR retinas compared to controls, whereas the pro-survival mitrochondrial protein BCL-xl was reduced. These alterations were largely blocked in the A2−/− OIR retina.

Conclusions

Our data implicate A2 in neurodegeneration during ROP. Deletion of A2 significantly improves neuronal survival and function, possibly through the regulation of mitochondrial membrane permeability mediated apoptosis during retinal ischemia. These molecular events are associated with decreased activation of glial cells, suggesting a rescue effect on macroglia as well.     

INFLUENCE OF FLORAL OPTICAL PROPERTIES ON THE ULTRAVIOLET RADIATION ENVIRONMENT OF POLLEN

Pollen in unopened flowers of most species is totally screened from solar ultraviolet-B radiation by imbricated petals that are largely opaque to UV-B. Following flower opening but before anther dehiscence, the anther walls of the species investigated filter out over 98% of the UV-B radiation. Reflectance of UV from corollas of open flowers does not generally appear to add significantly to the solar UV-B radiation environment of pollen.     

Active Nercc1 protein kinase concentrates at centrosomes early in mitosis and is necessary for proper spindle assembly

The Nercc1 protein kinase autoactivates in vitro and is activated in vivo during mitosis. Autoactivation in vitro requires phosphorylation of the activation loop at threonine 210. Mitotic activation of Nercc1 in mammalian cells is accompanied by Thr210 phosphorylation and involves a small fraction of total Nercc1. Mammalian Nercc1 coimmunoprecipitates gamma-tubulin and the activated Nercc1 polypeptides localize to the centrosomes and spindle poles during early mitosis, suggesting that active Nercc has important functions at the microtubular organizing center during cell division. To test this hypothesis, we characterized the Xenopus Nercc1 orthologue (XNercc). XNercc endogenous to meiotic egg extracts coprecipitates a multiprotein complex that contains gamma-tubulin and several components of the gamma-tubulin ring complex and localizes to the poles of spindles formed in vitro. Reciprocally, immunoprecipitates of the gamma-tubulin ring complex polypeptide Xgrip109 contain XNercc. Immunodepletion of XNercc from egg extracts results in delayed spindle assembly, fewer bipolar spindles, and the appearance of aberrant microtubule structures, aberrations corrected by addition of purified recombinant XNercc. XNercc immunodepletion also slows aster assembly induced by Ran-GTP, producing Ran-asters of abnormal size and morphology. Thus, Nercc1 contributes to both the centrosomal and the chromatin/Ran pathways that collaborate in the organization of a bipolar spindle.     

Crystal structures of human HSP90alpha-complexed with dihydroxyphenylpyrazoles

A series of dihydroxyphenylpyrazole compounds were identified as a unique class of reversible Hsp90 inhibitors. The crystal structures for two of the identified compounds complexed with the N-terminal ATP binding domain of human Hsp90alpha were determined. The dihydroxyphenyl ring of the compounds fits deeply into the adenine binding pocket with the C2 hydroxyl group forming a direct hydrogen bond with the side chain of Asp93. The pyrazole ring forms hydrogen bonds to the backbone carbonyl of Gly97, the hydroxyl group of Thr184 and to a water molecule, which is present in all of the published HSP90 structures. One of the identified compounds (G3130) demonstrated cellular activities (in Her-2 degradation and activation of Hsp70 promoter) consistent with the inhibition of cellular Hsp90 functions.     

Sequence polymorphism in polyploid wheat and their d-genome diploid ancestor

Sequencing was used to investigate the origin of the D genome of the allopolyploid species Triticum aestivum and Aegilops cylindrica. A 247-bp region of the wheat D-genome Xwye838 locus, encoding ADP-glucopyrophosphorylase, and a 326-bp region of the wheat D-genome Gss locus, encoding granule-bound starch synthase, were sequenced in a total 564 lines of hexaploid wheat (T. aestivum, genome AABBDD) involving all its subspecies and 203 lines of Aegilops tauschii, the diploid source of the wheat D genome. In Ae. tauschii, two SNP variants were detected at the Xwye838 locus and 11 haplotypes at the Gss locus. Two haplotypes with contrasting frequencies were found at each locus in wheat. Both wheat Xwye838 variants, but only one of the Gss haplotypes seen in wheat, were found among the Ae. tauschii lines. The other wheat Gss haplotype was not found in either Ae. tauschii or 70 lines of tetraploid Ae. cylindrica (genomes CCDD), which is known to hybridize with wheat. It is concluded that both T. aestivum and Ae. cylindrica originated recurrently, with at least two genetically distinct progenitors contributing to the formation of the D genome in both species.