Amber N. Juba

Oxidative stress and mitochondrial dysfunction are heavily implicated in all forms of Parkinson’s disease; however, antioxidant administration has largely failed in clinical trials. Among the likely causes of failure are brain bioavailability and cellular redox state. We have administered two manganese porphyrin compounds with different bioavailability, MnTE-2-PyP⁵⁺ and MnTnBuOE-2-PyP⁵⁺, to parkin-null Drosophila food and found that the more bioavailable one, with higher brain and mitochondrial availability, MnTnBuOE-2-PyP⁵⁺, improves developmental deficits and motivated behavior in female flies. Using highly sensitive redox reporters, we further found that MnTnBuOE-2-PyP⁵⁺ reduces hydrogen peroxide levels in mitochondria of dopaminergic neurons that are functionally homologous to the mammalian substantia nigra and facilitates motivated behavior in female flies. Interestingly, both compounds reduce an oxidative stress marker at the whole-brain level and extend lifespan in control flies. Neither compound improves lifespan in parkin-null flies. Thus, additional studies, changing the timing and/or dosage of compound administration, are warranted.

Loss-of-function parkin mutations cause oxidative stress and degeneration of dopaminergic neurons in the substantia nigra. Several consequences of parkin mutations have been described; to what degree they contribute to selective neurodegeneration remains unclear. Specific factors initiating excessive reactive oxygen species production, inefficient antioxidant capacity, or a combination are elusive. Identifying key oxidative stress contributors could inform targeted therapy. The absence of Drosophila parkin causes selective degeneration of a dopaminergic neuron cluster that is functionally homologous to the substantia nigra. By comparing observations in these to similar non-degenerating neurons, we may begin to understand mechanisms by which parkin loss of function causes selective degeneration. Using mitochondrially targeted redox-sensitive GFP2 fused with redox enzymes, we observed a sustained increased mitochondrial hydrogen peroxide levels in vulnerable dopaminergic neurons of parkin-null flies. Only transient increases in hydrogen peroxide were observed in similar but non-degenerating neurons. Glutathione redox equilibrium is preferentially dysregulated in vulnerable neuron mitochondria. To shed light on whether dysregulated glutathione redox equilibrium primarily contributes to oxidative stress, we supplemented food with folic acid, which can increase cysteine and glutathione levels. Folic acid improved survival, climbing, and transiently decreased hydrogen peroxide and glutathione redox equilibrium but did not mitigate whole-brain oxidative stress.

Since the successful use of vaccinia virus (VACV) in the immunization strategies to eliminate smallpox, research has been focused on the development of recombinant VACV strains expressing proteins from various pathogens. Attempts at decreasing the side effects associated with exposure to recombinant, wild-type viral strains have led to the development of attenuated viruses. Yet while these attenuated VACV’s have improved safety profiles compared to unmodified strains, their clinical use has been hindered due to efficacy issues in stimulating a host immune response. This deficiency has largely been attributed to decreased production of the target protein for immunization. Efforts to increase protein production from attenuated VACV strains has largely centered around modulation of viral factors, while manipulation of the translation of viral mRNAs has been largely unexplored. In this study we evaluate the use of translation enhancing element hTEE-658 to increase recombinant protein production in an attenuated VACV system. Optimization of the use of this motif is also attempted by combining it with strategies that have demonstrated effectiveness in previous research. We show that extension of the 5′ leader sequence containing hTEE-658 does not improve motif function, nor does the combination with other known translation enhancing elements. However, the sole use of hTEE-658 in an attenuated VACV system is shown to increase protein expression levels beyond those of a standard viral promoter when used with a wild-type virus. Taken together these results highlight the potential for hTEE-658 to improve the effectiveness of attenuated VACV vaccine candidates and give insights into the optimal sequence context for its use in vaccine design.

The classical model for eukaryotic translation initiation involves linear scanning, a cap‐dependent process in which the ribosome recognizes a 7‐methylguanosinecap at the 5′ end of the RNA message and scans the 5′ untranslated region for an appropriate initiation site. Alternatively, a growing body of evidence suggests that cap‐independent mechanisms play an important role during many cellular events where cap‐dependent scanning is inhibited. However despite this evidence, the mechanism by which this cap‐independent mode of translation occurs remains largely unknown. In a previous study, we began to address this by combining messenger RNA display with next‐generation deep sequencing to profile the human genome for RNA leader sequences that can enhance the translation of uncapped mRNA transcripts. Analysis of these sequences revealeda short, 13 nucleotide motif that was present in many of the RNAs isolated via this method. Given these findings, we propose that the identified 13 nucleotidemotif, when present within the leader sequence of an open reading frame, has an integral role in determining the increased translational activity driven by the larger leader sequence. To explore this role we examined the translation enhancing activity of several human in vitro selected translation enhancing elements (TEEs), which either contained or did not contain the 13 nucleotide motif. A series of reporter vectors containing the TEEs were used to assay the effect of the presence and absence of the motif on overall translation efficiency. Functional analysis studies performed in vitro and in cultured human and mammalian cells indicate that the 13 nucleotide motif, when present within a larger TEE, increased protein translation levels by several fold. Additional studies have identified distance constraints for translation enhancement with respect to the 13 nucleotide motif and the downstream open reading frame. Taken together these results provide evidence that the identified short 13 nucleotide motif plays an important role in the mechanism of cap‐independent translation from the human genome. This observation provides a new opportunity to explore the mechanistic details of human translation enhancing elements and their contribution to the human proteome. Support or Funding Information Funding for this research was provided by Midwestern University intramural funds awarded to BPW, a Midwestern University College of Veterinary Medicine research fellowship awarded to AH, and an NIH grant awarded to JCC