A Matrigel-based 3D construct of SH-SY5Y cells models the α-synuclein pathologies of Parkinson's disease

Parkinson's disease (PD) is featured with α-synuclein-based Lewy body pathology, which however was difficult to observe in conventional two-dimensional (2D) cell culture and even in animal models. We herein aimed to develop a three-dimensional (3D) cellular model of PD to recapitulate the α-synuclein pathologies. All-trans-retinoic acid-differentiated human SH-SY5Y cells and Matrigel were optimized for 3D construction. The 3D cultured cells displayed higher tyrosine hydroxylase expression and improved dopaminergic-like phenotypes than 2D cells as suggested by RNA-sequencing analyses. Multiple forms of α-synuclein, including monomer, low and high molecular weight oligomers, were differentially present in the 2D and 3D cells, but mostly remained unchanged upon the MPP+ or rotenone treatment. Phosphorylated α-synuclein was accumulated and detergent-insoluble α-synuclein fraction was observed in the neurotoxin-treated 3D cells. Importantly, Lewy body-like inclusions were captured in the 3D system, including proteinase K-resistant α-synuclein aggregates, ubiquitin aggregation, β-amyloid and β-sheet protein deposition. The study provides a unique and convenient 3D model of PD which recapitulates critical α-synuclein pathologies and should be useful in multiple PD-associated applications.

Protofibril formation: decreased total glutathione concentration as an early indicator of neuron damage in the brainstems of Wistar rats treated with rotenone

Background: Rotenone treatment causes oxidative stress in neurons and forms the basis of animal models of Parkinson's disease. The reduced form of glutathione is predicted to detoxify rotenone from neurons in the brainstem. This study aims to measure the concentration of total glutathione and analyze the formation of protofibril in the brainstem of Wistar rats treated with rotenone. Methods: Seventy-two male Wistar rats aged 8–9 weeks weighing 200–250 g were divided into two investigations: total glutathione determination and protofibril analysis. The independent variables were treatment group, observation time, and location in the brainstem. The dependent variables were the concentration of total glutathione and protofibril density. Results: The concentration of total glutathione was not significantly different among treatment groups (p: 0.084), observation time (p: 0.608), or the location in the brainstem (p: 0.372). Protofibril density was different in the treatment groups (p: 0.001), observation time (p: 0.001), and between the upper and lower brainstem (p: 0.001). Rotenone treatment subcortically induced the concentration of total glutathione in the brainstem to decrease, but protofibril density tended to increase. Conclusions: The total glutathione concentration is inversely proportional to protofibril density. Total glutathione might be an early marker of neuronal damage.

Rotenone Use and Subsequent Prey Loss Lowers Osprey Fledging Rates Via Brood Reduction

ABSTRACT Fisheries managers used the fish toxicant rotenone to eradicate an undesirable brown bullhead (Ameiurus nebulosus) population and all other fish species at Hyatt Reservoir, Oregon, on 12 October 1989. This 4-yr study (1988–1990, 1992) compared effects of that rotenone project on Ospreys (Pandion haliaetus) nesting at Hyatt Reservoir and nearby Howard Prairie Reservoir (untreated reference)—the latter a reservoir where both brown bullheads and hatchery-released rainbow trout (Oncorhynchus mykiss) prospered. Because Hyatt Reservoir was treated after Osprey fall migration in 1989, the first 2 yr (1988 and 1989) yielded pretreatment information: number of Osprey pairs was unchanged and reproductive rates were similar and consistent at the two reservoirs. Yearling fish (200–250 mm) were restocked at Hyatt Reservoir in the spring of 1990 and Ospreys returned each year following rotenone treatment, with no decline in the number of occupied or active nests. The negative effect of the rotenone treatment on Ospreys was short-term, resulting in reduced reproductive rates (young/occupied nest, young/active nest, and young/successful nest) during the first nesting season posttreatment, although hatching rates were not affected. Osprey dive success and prey delivery rates declined sharply in 1990, leading to competition for food among siblings and brood reduction. Osprey reproductive rates and prey delivery rates at Hyatt Reservoir in both 1990 and 1992 remained below the extremely high pretreatment rates, but within the range required for population stability. Serious adverse effects of the fish loss on Osprey reproduction were minimized by: (1) the delay of the rotenone application until after breeding season, (2) the restocking of the treated reservoir in the following spring with some larger (yearling) fish (though the timing was late), (3) the maintenance of a supplemental feeding program for a nesting pair of Bald Eagles (Haliaeetus leucocephalus), which minimized kleptoparasitism on Ospreys, and perhaps most important (4) the presence of nearby water bodies, where Osprey obtained some fish in the 1990 and 1992 breeding seasons.

Recovery of freshwater invertebrates in alpine lakes and streams following eradication of nonnative trout with rotenone

Nonnative fish eradication via the piscicide rotenone is an effective tool for fisheries management and conservation of native species. However, the long-term effects on non-target organisms, including benthic invertebrates and zooplankton in alpine lakes, are under-studied and are poorly understood. As part of a landscape-scale native fish conservation project, we assessed the effects of 50 ppb rotenone on the aquatic invertebrate community by comparing pre- and post-rotenone treatment density and diversity metrics of benthic invertebrates and zooplankton in 13 alpine lakes and their outlets in Montana, USA. Across study sites, decreases in density and diversity of some invertebrates, including Ephemeroptera, Plecoptera, and Trichoptera taxa, were observed the year following rotenone treatment, and within three years, densities and diversities were similar to and sometimes higher than pre-treatment values. These results demonstrate resilience of aquatic invertebrate communities following rotenone exposure in alpine lakes and streams and informs fisheries managers for planning rotenone projects and monitoring recovery of non-target organisms. Further studies will be useful to evaluate the mechanisms driving invertebrate recovery rates, including downstream drift from nontreated areas and terrestrial adult dispersal.

Ndfip1 Prevents Rotenone-Induced Neurotoxicity and Upregulation of α-Synuclein in SH-SY5Y Cells

Nedd4 family interacting protein 1 (Ndfip1) is an adaptor of Nedd4-family ubiquitin ligases. Experimental results showed that Ndfip1 had a potential neuroprotective effect in neurology diseases. However, the neuroprotective effect and the underlying mechanisms of Ndfip1 in Parkinson's disease (PD) have not yet been fully elucidated. Therefore, in this study, we explored the neuroprotective effect of Ndfip1 against mitochondrial complex I inhibitor rotenone in a human dopaminergic neuroblastoma SH-SY5Y cell line and further elucidated its possible underlying mechanisms. Our results showed that rotenone could induce the up-regulation of α-synuclein (α-syn) in both mRNA and protein levels. The expression of Ndfip1 decreased at 24 h after rotenone treatment. Further study showed that high expression of Ndfip1 could protect SH-SY5Y cells against rotenone-induced neurotoxicity and antagonize the rotenone-induced increase in α-syn protein levels. In addition, high expression of Ndfip1 inhibited rotenone-induced increase in the protein levels of caspase-3 and decrease in tyrosine hydroxylase (TH). Further study showed that Ndfip1 did not affect the protein expression of iron regulatory protein 1 (IRP1), transferrin receptor 1 (TfR1), while antagonized the increase in protein levels of P62 and ferritin L caused by rotenone. Our findings provide specific identification of Ndfip1 proteins to inhibit the increase of α-syn in rotenone-induced SH-SY5Y cells. Ndfip1 might be a new theoretical drug target for the prevention and treatment of PD.

Rapid Immunohistological Measurement of Tyrosine Hydroxylase in Rat Midbrain by Near-Infrared Instrument-Based Detection

We present a robust, fresh-frozen approach to immunohistochemistry (IHC), without committing the tissue to IHC via fixation and cryopreservation while maintaining long-term storage, using LiCor-based infrared (IR) quantification for sensitive assessment of TH in immunoreacted mid-brain sections for quantitative comparison across studies. In fresh-frozen tissue stored up to 1 year prior to IHC reaction, we found our method to be highly sensitive to rotenone treatment in 3-month-old Sprague-Dawley rats, and correlated with a significant decline in rotarod laten-cy-to-fall measurement by approximately 2.5 fold. The measured midbrain region revealed a 31% lower TH signal when compared to control (p<0.01 by t test, n=5). Bivariate analysis of in-tegrated TH counts versus rotarod latency-to-fall indicates a positive slope and modest but sig-nificant correlation of R2=0.68 (p<0.05, n=10). These results indicate this rapid, instrument-based quantification method by IR detection successfully quantifies TH levels in rat brain tissue, while taking only 5 days from euthanasia to data output. This approach also allows for the identifica-tion of multiple targets by IHC with the simultaneous performance of downstream molecular analysis within the same animal tissue, allowing for the use of fewer animals per study.

Aging Impairs Mitochondrial Function and Mitophagy and Elevates Interleukin 6 Within the Cerebral Vasculature

Background The blood‐brain barrier (BBB) is critical for cerebrovascular health. Although aging impairs the integrity of the BBB, the mechanisms behind this phenomenon are not clear. As mitochondrial components activate inflammation as mitochondria become dysfunctional, we examined how aging impacts cerebrovascular mitochondrial function, mitophagy, and inflammatory signaling; and whether any alterations correlate with BBB function. Methods and Results We isolated cerebral vessels from young (2–3 months of age) and aged (18–19 months of age) mice and found that aging led to increases in the cyclin‐dependent kinase inhibitor 1 senescence marker with impaired mitochondrial function, which correlated with aged mice exhibiting increased BBB leak compared with young mice. Cerebral vessels also exhibited increased expression of mitophagy proteins Parkin and Nix with aging. Using mitophagy reporter (mtKeima) mice, we found that the capacity to increase mitophagy from baseline within the cerebral vessels on rotenone treatment was reduced with aging. Aging within the cerebral vessels also led to the upregulation of the stimulator of interferon genes and increased interleukin 6 (IL‐6), a cytokine that alters mitochondrial function. Importantly, exogenous IL‐6 treatment of young cerebral vessels upregulated mitophagy and Parkin and impaired mitochondrial function; whereas inhibiting IL‐6 in aged cerebral vessels reduced Parkin expression and increased mitochondrial function. Furthermore, treating cerebral vessels of young mice with mitochondrial N‐formyl peptides upregulated IL‐6, increased Parkin, and reduced Claudin‐5, a tight junction protein integral to BBB integrity. Conclusions Aging alters the cerebral vasculature to impair mitochondrial function and mitophagy and increase IL‐6 levels. These alterations may impair BBB integrity and potentially reduce cerebrovascular health with aging.

INVESTIGATION OF THE NEUROPROTECTIVE EFFECT OF LINAGLIPTIN AND CELIPROLOL IN RESERPINE-INDUCED OROFACIAL DYSKINESIA AND ROTENONE-INDUCED NEURODEGENERATION IN RATS

Objective: Linagliptin, an anti-diabetic agent, proven to play an important role in regulating neuronal plasticity and reduce apoptosis and neuroinflammation by activating downstream AMPK/Sirt 1 pathway, which protects mitochondrial function and suppresses intracellular ROS accumulation and shows antioxidant action. Celiprolol, a β-1selective adrenoceptor blocker used as an anti-hypertensive agent, possesses a direct scavenging activity on oxygen radicals with antioxidant properties. The current study was designed to investigate the combined neuroprotective effect of linagliptin and celiprolol. Methods: Wistar rats of either sex were divided into different groups (n = 6). Eight groups each for Reserpine induced orofacial dyskinesia model and Rotenone induced neurodegeneration model to mimic Parkinson’s like conditions and treated or not with different doses of linagliptin and celiprolol. 24 h after the last dose, animals were subjected to behavioral, biochemical and histopathological evaluations. The data were analyzed by ANOVA and Bonferroni multiple comparison test. Results: Reserpine treatment increased VCMs, tongue protrusion and decreased locomotor activity. Rotenone treatment decreases the motor activity and exploratory ability of the animals. Reserpine as well as rotenone treatments decrease catalase, GSH, SOD and increase the LPO levels as compared to sham group animals. Reserpine and rotenone also showed the presence of ghost cells and vacuolated cytoplasm. Linagliptin and celiprolol alone as well as in combination normalized the behavioral, biochemical and histopathological complications. Conclusion: Linagliptin and Celiprolol showed neuroprotection by antioxidant activity as well as improved reserpine and rotenone-induced behavioral deficits. Both drugs have tenacious potential and can be used clinically with some further investigations.

Effects of Enteric Environmental Modification by Coffee Components on Neurodegeneration in Rotenone-Treated Mice

Epidemiological studies have shown that coffee consumption decreases the risk of Parkinson’s disease (PD). Caffeic acid (CA) and chlorogenic acid (CGA) are coffee components that have antioxidative properties. Rotenone, a mitochondrial complex I inhibitor, has been used to develop parkinsonian models, because the toxin induces PD-like pathology. Here, we examined the neuroprotective effects of CA and CGA against the rotenone-induced degeneration of central dopaminergic and peripheral enteric neurons. Male C57BL/6J mice were chronically administered rotenone (2.5 mg/kg/day), subcutaneously for four weeks. The animals were orally administered CA or CGA daily for 1 week before rotenone exposure and during the four weeks of rotenone treatment. Administrations of CA or CGA prevented rotenone-induced neurodegeneration of both nigral dopaminergic and intestinal enteric neurons. CA and CGA upregulated the antioxidative molecules, metallothionein (MT)-1,2, in striatal astrocytes of rotenone-injected mice. Primary cultured mesencephalic or enteric cells were pretreated with CA or CGA for 24 h, and then further co-treated with a low dose of rotenone (1–5 nM) for 48 h. The neuroprotective effects and MT upregulation induced by CA and CGA in vivo were reproduced in cultured cells. Our data indicated that intake of coffee components, CA and CGA, enhanced the antioxidative properties of glial cells and prevents rotenone-induced neurodegeneration in both the brain and myenteric plexus.