Results revealed that a higher fluorescent signal characterized the transduced cells Figure 2A.
Paraquat exposure indeed triggered a more robust DHE oxidation within cell bodies and neurites in the left DMnX, as assessed histologically as well as by quantification of the perikaryal fluorescent signal Figure 2, B and C. They were then treated with 2 i. They also received an injection of DHE 1 hour before the time of sacrifice. They also received a DHE injection. The number of Nissl-stained neurons was counted stereologically in the left DMnX. Box and whisker plots show median, upper and lower quartiles, and maximum and minimum as whiskers.
Furthermore, monoclonal antibodies i. NSyn24 labeling was sparse in the left AAV-injected side DMnX from saline-treated animals and revealed a neuritic, dot-like accumulation of the modified protein Figure 3A. PLA has previously been shown to enable visualization of proteins with specific posttranslational modifications, such as phosphorylation, SUMOylation, and acetylation 30 — Specific chromogenic dots were detected in the left but not the right DMnX of all AAV-injected mice, and a marked enhancement of signal characterized sections from paraquat- as compared with saline-treated animals Figure 3C and Supplemental Figure 3.
Squares highlight neuritic immunoreactivity, while the arrowhead indicates a DMnX immunoreactive cell body. CC, central canal. H Representative confocal images show left DMnX tissue. Box and whisker plots are shown. Results showed immunoreactivity within transduced cell bodies and neurites in the left DMnX of saline-injected mice. The extent of protein modifications detected by Syn was more pronounced, however, in animals treated with paraquat Figure 3H.
In particular, distinct chromogenic spots characterized the left DMnX ipsilateral to the AAV injection side , whereas no specific signal was detected on the contralateral side of the brain Figure 4C.
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B Representative confocal images of left DMnX tissue. Length and density of labeled fibers were then measured at the 7-day time point in a defined pontine region encompassing the locus coeruleus and parabrachial nucleus 20 , Both measurements showed values that were more than doubled as a result of paraquat administration Figure 5, C and D.
When fiber morphology was compared in sections from saline- versus paraquat-treated mice, these features were noticeably more prominent in the latter group of animals. Representative confocal images show labeled axons in the left pons. Prevention of paraquat-induced DMnX pathology.
A likely mechanism contributing to ROS production after paraquat exposure involves paraquat-microglia interactions. More specifically, microglial membrane-bound NADPH oxidase could catalyze the 1-electron reduction of paraquat, thus promoting its redox cycling with molecular oxygen and enhancing the formation of superoxide and other ROS 45 , Absence of this subunit prevents assembly of the enzyme at the plasma membrane and thus abolishes its catalytic activity 45 , Stereological counts of DMnX neurons were carried out to compare the neurodegenerative effects of paraquat administration in WT verus mutant mice Figure 7, C and D.
Quite remarkably, these effects were abolished in gp91phox-deficient mice. Gp91phox-deficient mice are resistant to paraquat-induced DMnX pathology. C Representative images of Nissl-stained neurons in the dorsal medulla oblongata. The left DMnX is delineated in red. In a set of initial experiments, V1S- and SV2-expressing cells were cocultured in the presence of vehicle or different concentrations of paraquat. Furthermore, total cell fluorescence caused by bimolecular complementation was measured and compared in randomly selected cells from vehicle- versus paraquat-treated cultures.
Further evidence of this association derived from experiments in which cocultures of V1S- and SV2-expressing cells were challenged with oxidative stress—inducing agents other than paraquat. In particular, the percentage of BiFC-positive cells was found to be more than doubled in cultures treated with hydrogen peroxide versus vehicle; it was also increased by approximately 5 times over control levels when hydrogen peroxide was added together with iron sulfate to the incubation medium Supplemental Figure 5.
Cell viability was measured and expressed as percentage of vehicle-treated cultures. Cell nuclei were stained with DAPI blue. PLA counts were divided by the number of cells, and values were averaged. The arrow indicates lack of signal colocalization, while the arrowheads show colocalization.
A control antibody, mouse IgG, was also used to account for nonspecific protein binding. IgG, anti—3-NT, or nSyn12 was added to cultures treated with either vehicle or paraquat. In vehicle-treated cultures, a comparison of the percentage of cells containing BiFC-positive aggregates after addition of IgG versus anti—3-NT or nSyn12 revealed decreased fluorescence in the presence of specific nitrated protein—binding antibodies; this decrease reached statistical significance with anti—3-NT, but not nSyn12, however Figure 8, J and K.
Oxidative challenges are likely to be common events in the CNS, underscoring the relevance of oxidative stress for pathogenetic processes in human neurodegenerative diseases. They can result from cellular dysfunction, such as mitochondrial impairment, and tissue reactions, such as activation of innate immunity 49 — In PD, oxidative stress could also be promoted by disease risk factors, including aging, environmental exposures, and genetic variants 22 , Here, we demonstrate a high susceptibility of DMnX neurons to oxidative stress; our data also indicate that these neurons are relatively more vulnerable to oxidative stress than other populations of cholinergic cells.
Following exposure to paraquat, DMnX neurons accumulated substantial amounts of ROS, whereas under the same toxic conditions, no apparent ROS buildup was detected within cholinergic cells in the nearby hypoglossal nucleus nor in the striatum and medial septal nucleus. This differential vulnerability, in spite of the same neurotransmitter phenotype, is not unique to cholinergic neurons, but is instead reminiscent of differences seen between midbrain dopaminergic cells. Dopaminergic neurons in the substantia nigra pars compacta are markedly more susceptible to oxidative challenges and ROS accumulation than dopaminergic cells in the adjacent ventral tegmental area 3 , 52 , The substantia nigra pars compacta is also known to be more vulnerable than the ventral tegmental area to the neurodegenerative process of PD 54 , Thus, taken together, previous and current observations support the likelihood that sensitivity to oxidative stress represents a predisposing factor common to PD-vulnerable neuronal populations.
The finding that both nigral and DMnX neurons display a high vulnerability to oxidative challenges is also noteworthy. A nigro-vagal pathway that controls gastric tone and motility has recently been shown to connect these 2 brain regions, raising the possibility that the pathological consequences of an oxidative injury may be relayed and possibly amplified through this anatomical and functional connection Nigral dopaminergic neurons and DMnX cholinergic cells share metabolic properties that could ultimately contribute to their high vulnerability to oxidative stress.
In particular, a prooxidant environment characterizing both nigral and DMnX neurons has been proposed to arise from their reliance on calcium currents for pacemaking firing activity. The high metabolic demands associated with cytosolic calcium oscillations could stimulate mitochondrial oxidative phosphorylation, increase mitochondrial ROS production, and predispose these neurons to ROS accumulation after oxidative challenges 4 , 11 , 50 , An important role of extraneuronal mechanisms in DMnX oxidative stress and ensuing pathology is indicated by our present results in transgenic mice lacking microglial NADPH oxidase activity.
Earlier investigations have indeed shown that, in models of nigrostriatal degeneration, lack of functional NADPH oxidase is associated with neuroprotection 45 , During oxidative stress, both superoxide anion and nitric oxide are likely to be generated. In particular, within neurons with calcium-dependent pacemaking, mitochondrial calcium influx has been shown to stimulate nitric oxide synthase activity, thus promoting nitric oxide production Reaction of nitric oxide with superoxide generates the peroxynitrite anion, which can readily dissociate into hydroxyl and nitrogen dioxide radicals; nitrogen dioxide directly reacts with tyrosine residues of proteins, resulting in their nitration to 3-NT Nitration-dependent changes in protein conformation may be stabilized by assembly into oligomers, thus contributing to this effect 38 , If it is assumed that accumulation of aggregate pathology plays a role in neuronal demise, then toxic oligomeric species would likely mediate this effect and contribute to the marked loss of DMnX neurons seen after severe oxidative stress.
Small protein aggregates may be accumulated and play a more relevant role at early stages of pathogenetic processes, including early damage after oxidative challenges 20 , Evidence strengthening this conclusion includes the findings of rescue experiments in which enhanced spreading by paraquat was virtually abolished in mice with reduced NADPH oxidase—dependent ROS generation.
In the in vitro setting, paraquat-induced increase in BiFC occurred in the absence of any overt evidence of cytotoxicity. Incubations in the presence of paraquat not only promoted BiFC, but also enhanced the extent of nitration of the fluorescent oligomers. Viral vectors. Gene expression was regulated by the human synapsin 1 promoter and enhanced using a woodchuck hepatitis virus posttranscriptional regulatory element and a polyadenylation signal sequence. Animals and treatment. Paraquat dichloride hydrate Sigma-Aldrich was dissolved in 0. Animals injected with 0. Mice were sacrificed with an i.
For analyses requiring nonfixed tissue e. Immunohistochemistry and image acquisition. A summary of primary antibodies and their source and working dilutions is shown in Supplemental Table 1. Single and double labeling were carried out for fluorescence and brightfield microscopy using established protocols with a few modifications 20 , Free-floating medulla oblongata sections for double-fluorescence staining were treated with an antigen-retrieval solution 10 mM sodium citrate in 0.
For nSyn Other mouse monoclonal antibodies were labeled by a 2-step protocol using first a horse anti-mouse biotinylated secondary antibody and then DyLight streptavidin All secondary antibodies were purchased from Vector Laboratories. Sections stained with nSyn A mouse brain atlas was used as reference for brain coordinates The total integrated density of the outlined puncta was quantified for each image, divided by the number of neurons, and averaged for each animal. For quantification of MDA, brains were removed and snap-frozen on dry ice.
Quantification of transduction. Investigators performing histological analyses were blinded to sample treatment. Every fifth section of the entire left DMnX between bregma —7. Counting of DMnX neurons. The number of Nissl-stained neurons was quantified throughout the entire DMnX using every fifth section. Coefficients of error were less than 0. Quantification of Syn fluorescence. Every fifth medulla oblongata section between bregma —7.
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