Neurotrophic factor MANF regulates autophagy and lysosome function to promote proteostasis in C. elegans.

The conserved mesencephalic astrocyte-derived neurotrophic factor (MANF) protects dopaminergic neurons but also functions in several other tissues. Previously, we showed that Caenorhabditis elegans manf-1 null mutants have increased ER stress, dopaminergic neurodegeneration, protein aggregation, slower growth, and a reduced lifespan. The multiple requirements of MANF in different systems suggest its essential role in regulating cellular processes. However, how intracellular and extracellular MANF regulates broader cellular function remains unknown. Here, we report a novel mechanism of action for manf-1 that involves the autophagy transcription factor HLH-30/TFEB-mediated signaling to regulate lysosomal function and aging. We generated multiple transgenic strains overexpressing MANF-1 and found that animals had extended lifespan, reduced protein aggregation, and improved neuronal health. Using a fluorescently tagged MANF-1, we observed different tissue localization of MANF-1 depending on the ER retention signal. Further subcellular analysis showed that MANF-1 localizes within cells to the lysosomes. These findings were consistent with our transcriptomic studies and, together with analysis of autophagy regulators, demonstrate that MANF-1 regulates protein homeostasis through increased autophagy and lysosomal activity. Collectively, our findings establish MANF as a critical regulator of the stress response, proteostasis, and aging.

Chronic high-sugar diet in adulthood protects Caenorhabditis elegans from 6-OHDA-induced dopaminergic neurodegeneration.

BACKGROUND: Diets high in saturated fat and sugar, termed "Western diets," have been associated with several negative health outcomes, including increased risk for neurodegenerative disease. Parkinson's disease (PD) is the second most prevalent neurodegenerative disease and is characterized by the progressive death of dopaminergic neurons in the brain. We build upon previous work characterizing the impact of high-sugar diets in Caenorhabditis elegans to mechanistically evaluate the relationship between high-sugar diets and dopaminergic neurodegeneration. RESULTS: Adult high-glucose and high-fructose diets, or exposure from day 1 to 5 of adulthood, led to increased lipid content, shorter lifespan, and decreased reproduction. However, in contrast to previous reports, we found that adult chronic high-glucose and high-fructose diets did not induce dopaminergic neurodegeneration alone and were protective from 6-hydroxydopamine (6-OHDA) induced degeneration. Neither sugar altered baseline electron transport chain function and both increased vulnerability to organism-wide ATP depletion when the electron transport chain was inhibited, arguing against energetic rescue as a basis for neuroprotection. The induction of oxidative stress by 6-OHDA is hypothesized to contribute to its pathology, and high-sugar diets prevented this increase in the soma of the dopaminergic neurons. However, we did not find increased expression of antioxidant enzymes or glutathione levels. Instead, we found evidence suggesting downregulation of the dopamine reuptake transporter dat-1 that could result in decreased 6-OHDA uptake. CONCLUSIONS: Our work uncovers a neuroprotective role for high-sugar diets, despite concomitant decreases in lifespan and reproduction. Our results support the broader finding that ATP depletion alone is insufficient to induce dopaminergic neurodegeneration, whereas increased neuronal oxidative stress may drive degeneration. Finally, our work highlights the importance of evaluating lifestyle by toxicant interactions.

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. This study reveals phenotypic consequences of whole-animal tetraploidy that make C. elegans an excellent model for ploidy differences.

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. Overall, this study reveals the phenotypic consequences of whole-animal tetraploidy in C. elegans.

Mild pentachlorophenol-mediated uncoupling of mitochondria depletes ATP but does not cause an oxidized redox state or dopaminergic neurodegeneration in Caenorhabditis elegans.

Aims: Mitochondrial dysfunction is implicated in several diseases, including neurological disorders such as Parkinson's disease. However, there is uncertainty about which of the many mechanisms by which mitochondrial function can be disrupted may lead to neurodegeneration. Pentachlorophenol (PCP) is an organic pollutant reported to cause mitochondrial dysfunction including oxidative stress and mitochondrial uncoupling. We investigated the effects of PCP exposure in Caenorhabditis elegans, including effects on mitochondria and dopaminergic neurons. We hypothesized that mild mitochondrial uncoupling by PCP would impair bioenergetics while decreasing oxidative stress, and therefore would not cause dopaminergic neurodegeneration. Results: A 48-hour developmental exposure to PCP causing mild growth delay (∼10 % decrease in growth during 48 h, covering all larval stages) reduced whole-organism ATP content > 50 %, and spare respiratory capacity âˆ¼ 30 %. Proton leak was also markedly increased. These findings suggest a main toxic mechanism of mitochondrial uncoupling rather than oxidative stress, which was further supported by a concomitant shift toward a more reduced cellular redox state measured at the whole organism level. However, exposure to PCP did not cause dopaminergic neurodegeneration, nor did it sensitize animals to a neurotoxic challenge with 6-hydroxydopamine. Whole-organism uptake and PCP metabolism measurements revealed low overall uptake of PCP in our experimental conditions (50 µM PCP in the liquid exposure medium resulted in organismal concentrations of < 0.25 µM), and no measurable production of the oxidative metabolites tetra-1,4-benzoquinone and tetrachloro-p-hydroquinone. Innovation: This study provides new insights into the mechanistic interplay between mitochondrial uncoupling, oxidative stress, and neurodegeneration in C. elegans. These findings support the premise of mild uncoupling-mediated neuroprotection, but are inconsistent with proposed broad "mitochondrial dysfunction"-mediated neurodegeneration models, and highlight the utility of the C. elegans model for studying mitochondrial and neurotoxicity. Conclusions: Developmental exposure to pentachlorophenol causes gross toxicological effects (growth delay and arrest) at high levels. At a lower level of exposure, still causing mild growth delay, we observed mitochondrial dysfunction including uncoupling and decreased ATP levels. However, this was associated with a more-reduced cellular redox tone and did not exacerbate dopaminergic neurotoxicity of 6-hydroxydopamine, instead trending toward protection. These findings may be informative of efforts to define nuanced mitochondrial dysfunction-related adverse outcome pathways that will differ depending on the form of initial mitochondrial toxicity.

Role of Mitochondrial Cytochrome P450 2E1 in Healthy and Diseased Liver.

Cytochrome P450 2E1 (CYP2E1) is pivotal in hepatotoxicity induced by alcohol abuse and different xenobiotics. In this setting, CYP2E1 generates reactive metabolites inducing oxidative stress, mitochondrial dysfunction and cell death. In addition, this enzyme appears to play a role in the progression of obesity-related fatty liver to nonalcoholic steatohepatitis. Indeed, increased CYP2E1 activity in nonalcoholic fatty liver disease (NAFLD) is deemed to induce reactive oxygen species overproduction, which in turn triggers oxidative stress, necroinflammation and fibrosis. In 1997, Avadhani's group reported for the first time the presence of CYP2E1 in rat liver mitochondria, and subsequent investigations by other groups confirmed that mitochondrial CYP2E1 (mtCYP2E1) could be found in different experimental models. In this review, we first recall the main features of CYP2E1 including its role in the biotransformation of endogenous and exogenous molecules, the regulation of its expression and activity and its involvement in different liver diseases. Then, we present the current knowledge on the physiological role of mtCYP2E1, its contribution to xenobiotic biotransformation as well as the mechanism and regulation of CYP2E1 targeting to mitochondria. Finally, we discuss experimental investigations suggesting that mtCYP2E1 could have a role in alcohol-associated liver disease, xenobiotic-induced hepatotoxicity and NAFLD.

Aging reduces liver resiliency by dysregulating Hedgehog signaling.

Older age is a major risk factor for damage to many tissues, including liver. Aging undermines resiliency and impairs liver regeneration. The mechanisms whereby aging reduces resiliency are poorly understood. Hedgehog is a signaling pathway with critical mitogenic and morphogenic functions during development. Recent studies indicate that Hedgehog regulates metabolic homeostasis in adult liver. The present study evaluates the hypothesis that Hedgehog signaling becomes dysregulated in hepatocytes during aging, resulting in decreased resiliency and therefore, impaired regeneration and enhanced vulnerability to damage. Partial hepatectomy (PH) was performed on young and old wild-type mice and Smoothened (Smo)-floxed mice treated with viral vectors to conditionally delete Smo and disrupt Hedgehog signaling specifically in hepatocytes. Changes in signaling were correlated with changes in regenerative responses and compared among groups. Old livers had fewer hepatocytes proliferating after PH. RNA sequencing identified Hedgehog as a top downregulated pathway in old hepatocytes before and after the regenerative challenge. Deleting Smo in young hepatocytes before PH prevented Hedgehog pathway activation after PH and inhibited regeneration. Gene Ontogeny analysis demonstrated that both old and Smo-deleted young hepatocytes had activation of pathways involved in innate immune responses and suppression of several signaling pathways that control liver growth and metabolism. Hedgehog inhibition promoted telomere shortening and mitochondrial dysfunction in hepatocytes, consequences of aging that promote inflammation and impair tissue growth and metabolic homeostasis. Hedgehog signaling is dysregulated in old hepatocytes. This accelerates aging, resulting in decreased resiliency and therefore, impaired liver regeneration and enhanced vulnerability to damage.

PCR-Based Determination of Mitochondrial DNA Copy Number in Multiple Species.

Mitochondrial DNA (mtDNA) copy number is a critical component of overall mitochondrial health. In this chapter, we describe methods for simultaneous isolation of mtDNA and nuclear DNA (nucDNA), and measurement of their respective copy numbers using quantitative PCR. Methods differ depending on the species and cell type of the starting material, and availability of specific PCR reagents. We also briefly describe factors that affect mtDNA copy number and discuss caveats to its use as a biomarker.

Recent developments in in vitro and in vivo models for improved translation of preclinical pharmacokinetics and pharmacodynamics data.

Improved pharmacokinetics/pharmacodynamics (PK/PD) prediction in the early stages of drug development is essential to inform lead optimization strategies and reduce attrition rates. Recently, there have been significant advancements in the development of new in vitro and in vivo strategies to better characterize pharmacokinetic properties and efficacy of drug leads. Herein, we review advances in experimental and mathematical models for clearance predictions, advancements in developing novel tools to capture slowly metabolized drugs, in vivo model developments to capture human etiology for supporting drug development, limitations and gaps in these efforts, and a perspective on the future in the field.

Xenobiotic metabolism and transport in Caenorhabditis elegans.

Caenorhabditis elegans has emerged as a major model in biomedical and environmental toxicology. Numerous papers on toxicology and pharmacology in C. elegans have been published, and this species has now been adopted by investigators in academic toxicology, pharmacology, and drug discovery labs. C. elegans has also attracted the interest of governmental regulatory agencies charged with evaluating the safety of chemicals. However, a major, fundamental aspect of toxicological science remains underdeveloped in C. elegans: xenobiotic metabolism and transport processes that are critical to understanding toxicokinetics and toxicodynamics, and extrapolation to other species. The aim of this review was to initially briefly describe the history and trajectory of the use of C. elegans in toxicological and pharmacological studies. Subsequently, physical barriers to chemical uptake and the role of the worm microbiome in xenobiotic transformation were described. Then a review of what is and is not known regarding the classic Phase I, Phase II, and Phase III processes was performed. In addition, the following were discussed (1) regulation of xenobiotic metabolism; (2) review of published toxicokinetics for specific chemicals; and (3) genetic diversity of these processes in C. elegans. Finally, worm xenobiotic transport and metabolism was placed in an evolutionary context; key areas for future research highlighted; and implications for extrapolating C. elegans toxicity results to other species discussed.

Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

Advances in the study of drug metabolism - symposium report of the 12th Meeting of the International Society for the Study of Xenobiotics (ISSX).

The 12th International Society for the Study of Xenobiotics (ISSX) meeting, held in Portland, OR, USA from July 28 to 31, 2019, was attended by diverse members of the pharmaceutical sciences community. The ISSX New Investigators Group provides learning and professional growth opportunities for student and early career members of ISSX. To share meeting content with those who were unable to attend, the ISSX New Investigators herein elected to highlight the "Advances in the Study of Drug Metabolism" symposium, as it engaged attendees with diverse backgrounds. This session covered a wide range of current topics in drug metabolism research including predicting sites and routes of metabolism, metabolite identification, ligand docking, and medicinal and natural products chemistry, and highlighted approaches complemented by computational modeling. In silico tools have been increasingly applied in both academic and industrial settings, alongside traditional and evolving in vitro techniques, to strengthen and streamline pharmaceutical research. Approaches such as quantum mechanics simulations facilitate understanding of reaction energetics toward prediction of routes and sites of drug metabolism. Furthermore, in tandem with crystallographic and orthogonal wet lab techniques for structural validation of drug metabolizing enzymes, in silico models can aid understanding of substrate recognition by particular enzymes, identify metabolic soft spots and predict toxic metabolites for improved molecular design. Of note, integration of chemical synthesis and biosynthesis using natural products remains an important approach for identifying new chemical scaffolds in drug discovery. These subjects, compiled by the symposium organizers, presenters, and the ISSX New Investigators Group, are discussed in this review.

Zebrafish CYP1A expression in transgenic Caenorhabditis elegans protects from exposures to benzo[a]pyrene and a complex polycyclic aromatic hydrocarbon mixture.

Polycyclic aromatic hydrocarbons (PAHs) are environmental toxicants primarily produced during incomplete combustion; some are carcinogens. PAHs can be safely metabolized or, paradoxically, bioactivated via specific cytochrome P450 (CYP) enzymes to more reactive metabolites, some of which can damage DNA and proteins. Among the CYP isoforms implicated in PAH metabolism, CYP1A enzymes have been reported to both sensitize and protect from PAH toxicity. To clarify the role of CYP1A in PAH toxicity, we generated transgenic Caenorhabditis elegans that express CYP1A at a basal (but not inducible) level. Because this species does not normally express any CYP1 family enzyme, this approach permitted a test of the role of basally expressed CYP1A in PAH toxicity. We exposed C. elegans at different life stages to either the PAH benzo[a]pyrene (BaP) alone, or a real-world mixture dominated by PAHs extracted from the sediment of a highly contaminated site on the Elizabeth River (VA, USA). This site, the former Atlantic Wood Industries, was declared a Superfund site due to coal tar creosote contamination that caused very high levels (in the [mg/mL] range) of high molecular weight PAHs within the sediments. We demonstrate that CYP1A protects against BaP-induced growth delay, reproductive toxicity, and reduction of steady state ATP levels. Lack of sensitivity of a DNA repair (Nucleotide Excision Repair)-deficient strain suggested that CYP1A did not produce significant levels of DNA-reactive metabolites from BaP. The protective effects of CYP1A in Elizabeth River sediment extract (ERSE)-exposed nematodes were less pronounced than those seen in BaP-exposed nematodes; CYP1A expression protected against ERSE-induced reduction of steady-state ATP levels, but not other outcomes of exposure to sediment extracts. Overall, we find that in C. elegans, a basal level of CYP1A activity is protective against the examined PAH exposures.

Fluorescence-based sorting of Caenorhabditis elegans via acoustofluidics.

Effectively isolating and categorizing large quantities of Caenorhabditis elegans (C. elegans) based on different phenotypes is important for most worm research, especially genetics. Here we present an integrated acoustofluidic chip capable of identifying worms of interest based on expression of a fluorescent protein in a continuous flow and then separate them accordingly in a high-throughput manner. Utilizing planar fiber optics as the detection unit, our acoustofluidic device requires no temporary immobilization of worms for interrogation/detection, thereby improving the throughput. Implementing surface acoustic waves (SAW) as the sorting unit, our device provides a contact-free method to move worms of interest to the desired outlet, thus ensuring the biocompatibility for our chip. Our device can sort worms of different developmental stages (L3 and L4 stage worms) at high throughput and accuracy. For example, L3 worms can be processed at a throughput of around 70 worms per min with a sample purity over 99%, which remains over 90% when the throughput is increased to around 115 worms per min. In our acoustofluidic chip, the time period to complete the detection and sorting of one worm is only 50 ms, which outperforms nearly all existing microfluidics-based worm sorting devices and may be further reduced to achieve higher throughput.

In Vitro Metabolism of Isopropylated and tert-Butylated Triarylphosphate Esters Using Human Liver Subcellular Fractions.

Isopropylated and tert-butylated triarylphosphate esters (ITPs and TBPPs, respectively) are plasticizers and flame retardants that are ubiquitous in indoor environments; however, no studies to date have characterized their metabolism. Using human liver subcellular S9 fractions, phase I and II in vitro metabolism of triphenyl phosphate (TPHP), 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), 2-isopropylphenyl diphenyl phosphate (2IPPDPP), and 4-isopropylphenyl diphenyl phosphate (4IPPDPP) was investigated at 1 and 10 µM doses. Parent depletion and the formation of known or suspected metabolites (e.g., likely hydrolysis or hydroxylated products), including diphenyl phosphate (DPHP), hydroxyl-triphenyl phosphate (OH-TPHP), isopropylphenyl phenyl phosphate (ip-PPP), and tert-butylphenyl phenyl phosphate (tb-PPP), were monitored and quantified via GC/MS or LC-MS/MS. tb-PPP and its conjugates were identified as the major in vitro metabolites of 4tBPDPP and accounted for 71% and 49%, respectively, of the parent molecule that was metabolized during the incubation. While the mass balance between parents and metabolites was conserved for TPHP and 4tBPDPP, approximately 20% of the initial parent mass was unaccounted for after quantifying suspected metabolites of 2IPPDPP and 4IPPDPP that had authentic standards available. Two novel ITP metabolites, mono-isopropenylphenyl diphenyl phosphate and hydroxy-isopropylphenyl diphenyl phosphate, were tentatively identified by high-resolution mass spectrometry and screened for in recently collected human urine where mono-isopropenylphenyl diphenyl phosphate was detected in one of nine samples analyzed. This study provides insight into the biological fate of ITP and TBPP isomers in human tissues and is useful in identifying appropriate biomarkers of exposure to monitor, particularly in support of epidemiological studies.

Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration.

Regular physical exercise is the most efficient and accessible intervention known to promote healthy aging in humans. The molecular and cellular mechanisms that mediate system-wide exercise benefits, however, remain poorly understood, especially as applies to tissues that do not participate directly in training activity. The establishment of exercise protocols for short-lived genetic models will be critical for deciphering fundamental mechanisms of transtissue exercise benefits to healthy aging. Here we document optimization of a long-term swim exercise protocol for Caenorhabditis elegans and we demonstrate its benefits to diverse aging tissues, even if exercise occurs only during a restricted phase of adulthood. We found that multiple daily swim sessions are essential for exercise adaptation, leading to body wall muscle improvements in structural gene expression, locomotory performance, and mitochondrial morphology. Swim exercise training enhances whole-animal health parameters, such as mitochondrial respiration and midlife survival, increases functional healthspan of the pharynx and intestine, and enhances nervous system health by increasing learning ability and protecting against neurodegeneration in models of tauopathy, Alzheimer's disease, and Huntington's disease. Remarkably, swim training only during early adulthood induces long-lasting systemic benefits that in several cases are still detectable well into midlife. Our data reveal the broad impact of swim exercise in promoting extended healthspan of multiple C. elegans tissues, underscore the potency of early exercise experience to influence long-term health, and establish the foundation for exploiting the powerful advantages of this genetic model for the dissection of the exercise-dependent molecular circuitry that confers system-wide health benefits to aging adults.

Strengths and limitations of morphological and behavioral analyses in detecting dopaminergic deficiency in Caenorhabditis elegans.

In order to develop a better understanding of the role environmental toxicants may play in the onset and progression of neurodegenerative diseases, it has become increasingly important to optimize sensitive methods for quickly screening toxicants to determine their ability to disrupt neuronal function. The nematode Caenorhabditis elegans can help with this effort. This species has an integrated nervous system producing behavioral function, provides easy access for molecular studies, has a rapid lifespan, and is an inexpensive model. This study focuses on methods of measuring neurodegeneration involving the dopaminergic system and the identification of compounds with actions that disrupt dopamine function in the model organism C. elegans. Several dopamine-mediated locomotory behaviors, Area Exploration, Body Bends, and Reversals, as well as Swimming-Induced Paralysis and Learned 2-Nonanone Avoidance, were compared to determine the best behavioral method for screening purposes. These behavioral endpoints were also compared to morphological scoring of neurodegeneration in the dopamine neurons. We found that in adult worms, Area Exploration is more advantageous than the other behavioral methods for identifying DA-deficient locomotion and is comparable to neuromorphological scoring outputs. For larval stage worms, locomotion was an unreliable endpoint, and neuronal scoring appeared to be the best method. We compared the wild-type N2 strain to the commonly used dat-1p::GFP reporter strains BY200 and BZ555, and we further characterized the dopamine-deficient strains, cat-2 e1112 and cat-2 n4547. In contrast to published results, we found that the cat-2 strains slowed on food almost as much as N2s. Both showed decreased levels of cat-2 mRNA and DA content, rather than none, with cat-2 e1112 having the greatest reduction in DA content in comparison to N2. Finally, we compared and contrasted strengths, limitations, cost, and equipment needs for all primary methods for analysis of the dopamine system in C. elegans.

Genetic Defects in Mitochondrial Dynamics in Caenorhabditis elegans Impact Ultraviolet C Radiation- and 6-hydroxydopamine-Induced Neurodegeneration.

BACKGROUND: Parkinson's disease (PD) is one of the most common neurodegenerative disorders involving devastating loss of dopaminergic neurons in the substantia nigra. Early steps in PD pathogenesis include mitochondrial dysfunction, and mutations in mitochondrial genes have been linked to familial forms of the disease. However, low penetrance of mutations indicates a likely important role for environmental factors in PD risk through gene by environment interactions. Herein, we study how genetic deficiencies in mitochondrial dynamics processes including fission, fusion, and mitophagy interact with environmental exposures to impact neurodegeneration. METHODS: We utilized the powerful model organism Caenorhabditis elegans to study ultraviolet C radiation (UVC)- and 6-hydroxydopamine-induced degeneration of fluorescently-tagged dopaminergic neurons in the background of fusion deficiency (MFN1/2 homolog, fzo-1), fission deficiency (DMN1L homolog, drp-1), and mitochondria-specific autophagy (mitophagy) deficiency (PINK1 and PRKN homologs, pink-1 and pdr-1). RESULTS: Overall, we found that deficiency in either mitochondrial fusion or fission sensitizes nematodes to UVC exposure (used to model common environmental pollutants) but protects from 6-hydroxydopamine-induced neurodegeneration. By contrast, mitophagy deficiency makes animals more sensitive to these stressors with an interesting exception-pink-1 deficiency conferred remarkable protection from 6-hydroxydopamine. We found that this protection could not be explained by compensatory antioxidant gene expression in pink-1 mutants or by differences in mitochondrial morphology. CONCLUSIONS: Together, our results support a strong role for gene by environment interactions in driving dopaminergic neurodegeneration and suggest that genetic deficiency in mitochondrial processes can have complex effects on neurodegeneration.

Open source acoustofluidics.

Over the past several decades, a litany of acoustofluidic devices have been developed which purport to have significant advantages over traditional benchtop analytical tools. These acoustofluidic devices are frequently labeled as "labs-on-chips"; however, many do an insufficient job of limiting their dependence on the lab. Often, acoustofluidic devices still require skilled operators and complex external equipment. In an effort to address these shortcomings, we developed a low-cost, expandable, and multifunctional system for controlling acoustofluidic devices in the audible to low ultrasonic frequency range (31 Hz to 65 kHz). The system was designed around the readily available Arduino prototyping platform because of its user-friendly coding environment and expansive network of open source material; these factors enabled us to create a system capable of generating high voltage oscillatory signals and controlling microscale flows in acoustofluidic devices. Utilizing the established open source system, we achieved a series of acoustofluidic applications involving the manipulation of fluids and biological objects in a portable fashion. In particular, we used our open source acoustofluidic devices to achieve active rotation of cells and microorganisms, and operation of an acoustofluidic mixing device which has previously shown potential for viscous sample preparation, in a portable fashion. Additionally, using low frequency flexural waves and our portable system, we achieved acoustofluidic separation of particles based on size. It is our hope that the open source platform presented here can pave the way for future acoustofluidic devices to be used at the point-of-care, as well as simplify the operation of these devices to enable resource limited users to leverage the benefits of acoustofluidics in their work.

MANF deletion abrogates early larval Caenorhabditis elegans stress response to tunicamycin and Pseudomonas aeruginosa.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is the only human neurotrophic factor with an evolutionarily-conserved C. elegans homolog, Y54G2A.23 or manf-1. MANF is a small, soluble, endoplasmic-reticulum (ER)-resident protein that is secreted upon ER stress and promotes survival of target cells such as neurons. However, the role of MANF in ER stress and its mechanism of cellular protection are not clear and the function of MANF in C. elegans is only beginning to emerge. In this study, we show that depletion of C. elegans manf-1 causes a slight decrease in lifespan and brood size; furthermore, combined depletion of manf-1 and the IRE-1/XBP-1 ER stress/UPR pathway resulted in sterile animals that did not produce viable progeny. We demonstrate upregulation of markers of ER stress in L1 larval nematodes, as measured by hsp-3 and hsp-4 transcription, upon depletion of manf-1 by RNAi or mutation; however, there was no difference in tunicamycin-induced expression of hsp-3 and hsp-4 between wild-type and MANF-deficient worms. Surprisingly, larval growth arrest observed in wild-type nematodes reared on tunicamycin is completely prevented in the manf-1 (tm3603) mutant. Transcriptional microarray analysis revealed that manf-1 mutant L1 larvae exhibit a novel modulation of innate immunity genes in response to tunicamycin. The hypothesis that manf-1 negatively regulates the innate immunity pathway is supported by our finding that the development of manf-1 mutant larvae compared to wild-type larvae is not inhibited by growth on P. aeruginosa. Together, our data represent the first characterization of C. elegans MANF as a key modulator of organismal ER stress and immunity.