PFAS soil concentrations surrounding a hazardous waste incinerator in East Liverpool, Ohio, an environmental justice community.

Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic compounds widely used in industrial and consumer products. While PFAS provide product durability, these chemicals are ubiquitous, persistent, bioaccumulative, and toxic. These characteristics make the ultimate disposal of PFAS a challenge. One current disposal method is incineration; however, little research has been conducted on the safety and effectiveness of PFAS incineration. The characteristics of communities with hazardous waste incinerators that have received PFAS shipments indicate that more individuals with lower incomes and individuals with less education than the US average are at higher risk of exposure, which presents important environmental justice and health equity concerns of PFAS incineration. Situated in eastern Ohio, East Liverpool is an Appalachian community that is home to a large hazardous-waste incinerator, operated by Heritage WTI, that began accepting PFAS in 2019. Residents are concerned that the disposal lacks the research necessary to assure safety for the residents. Due to both community interest and data gaps regarding PFAS incineration, our research team conducted a pilot study to examine the distribution and concentration of PFAS in soil samples surrounding the incinerator. All 35 soil samples had measurable amounts of PFAS including perfluorobutanesulfonic acid (PFBS), perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and hexafluoropropylene oxide dimer acid (HFPO-DA)/GenX. PFOS was measured in the majority of soil samples (97%) with a range of 50-8,300 ng/kg. PFOA was measured in 94% of soil samples with a range of 51 ng/kg to 1300 ng/kg. HFPO-DA/GenX was measurable in 12 soil samples with concentrations of ranging from 150 ng/kg to 1500 ng/kg. Further research on PFAS disposal will advance knowledge and action related to regulatory requirements and exposure prevention, ultimately improving individual and community protections and health equity.

Direct injection analysis of per and polyfluoroalkyl substances in surface and drinking water by sample filtration and liquid chromatography-tandem mass spectrometry.

We developed and validated a method for direct determination of per- and polyfluoroalkylated substances (PFASs) in environmental water samples without prior sample concentration. Samples are centrifuged and supernatants passed through an Acrodisc Filter (GXF/GHP 0.2  um, 25  mm diameter). After addition of ammonium acetate, samples are analyzed by UPLC-MS/MS using an AB Sciex 6500 plus Q-Trap mass spectrometer operated in negative multiple reaction-monitoring (MRM) mode. The instrument system incorporates a delay column between the pumps and autosampler to mitigate interference from background PFAS. The method monitors eight short-/long-chain PFAS which are identified by monitoring specific precursor product ion pairs and by their retention times and quantified using isotope mass-labeled internal standard based calibration plots. Average spiked recoveries (n = 8) of target analytes ranged from 84 to 110% with 4-9% relative standard deviation (RSD). The mean spiked recoveries (n = 8) of four surrogates were 94-106% with 3-8% RSD. For continuous calibration verification (CCV), average spiked recoveries (n = 8) for target analytes ranged from 88 to 114% with 4-11% RSD and for surrogates ranged from 104-112% with 3-11% RSD. The recoveries (n = 6) of matrix spike (MX), matrix spike duplicate (MXD), and field reagent blank (FRB) met our acceptance criteria. The limit of detection for the target analytes was between 0.007 and 0.04 ng/mL. The method was used to measure PFAS in tap water and surface water.

Interplay between protein synthesis and degradation in the CNS: physiological and pathological implications.

Compromise of the ubiquitin-proteasome system (UPS) is a potential basis for multiple physiological abnormalities and pathologies in the CNS. This could be because reduced protein turnover leads to bulk intracellular protein accumulation. However, conditions associated with compromised UPS function are also associated with impairments in protein synthesis, and impairment of UPS function is sufficient to inhibit protein synthesis. These data suggest that the toxicity of UPS inhibition need not depend on gross intracellular protein accumulation, and indicate the potential for crosstalk between the UPS and protein-synthesis pathways. In this review, we discuss evidence for interplay between the UPS and protein-synthesis machinery, and outline the implications of this crosstalk for physiological and pathological processes in the CNS.

Cell-specific actions of HIV-Tat and morphine on opioid receptor expression in glia.

HIV-1 patients who abuse opiate-based drugs, including heroin and morphine, are at a higher risk of developing HIV dementia. The effects of opiates are mediated predominantly through opioid receptors, which are expressed on glial cells. As HIV-1 infection in the CNS is restricted to glial cells, experiments were designed to measure the cell-specific effects of HIV Tat and morphine exposure on opioid receptor expression in both astrocytes and microglia. Specifically, the cell-type-specific pattern of mu opioid receptor (MOR), delta opioid receptor (DOR), and kappa opioid receptor (KOR) localization (surface vs. intracellular) and expression of opioid receptor mRNA were determined after exposure to morphine in the presence and the absence of Tat in primary cultured microglia and astrocytes. Data show that morphine treatment caused significantly decreased cell surface expression of opioid receptors in microglia but not in astrocytes. However, morphine treatment in the presence of Tat significantly increased intracellular expression of opioid receptors and prevented morphine-induced cell surface opioid receptor down-regulation in microglia. These findings document that cell surface opioid receptor expression is divergently regulated by morphine in microglia compared with in astrocytes, and further suggest that HIV-Tat could exacerbate opioid receptor signaling in microglia by increasing receptor expression and/or altering ligand-induced trafficking of opioid receptors.

Upregulation of PSMB8 and cathepsins in the human brains of dementia with Lewy bodies.

Proteasome and lysosome are responsible for the homeostasis of proteins, lipids and carbohydrates in cells. Numerous reports indicate the proteolytic pathways have altered functions during neurodegeneration and aging. Dementia with Lewy bodies (DLB) is one of the leading forms of dementia, and the proteolytic alteration in DLB has not yet been fully investigated. This study shows that the components of proteasome and lysosome had selectively altered gene expression and enzymatic functions. Specifically, PSMB8, an inducible proteasomal ß subunit, had elevated mRNA level and protein level in DLB brain compared with age-matched controls. The proteasomal caspase-like peptidase showed significant decreased activity in DLB brains and the trypsin-like/chemotrypsin-like activities did not reach statistical significance. Lysosomal cathepsin B and D had elevated mRNA levels while only cathepsin B showed elevated enzymatic activity in DLB brains. This data indicate that the alteration of proteolytic pathways is highly selective and comprehensive. Further study to elucidate the correlation between neurodegenerative development and the alteration of proteolytic pathways would be important for therapeutic development.

Proteasome inhibition induces reversible impairments in protein synthesis.

Proteasome inhibition occurs during normal aging and in a variety of age-related diseases, with inhibition of proteasome function sufficient to induce physiological and pathological alterations observed in each of these conditions. It is presumed that proteasome inhibition induces cellular alterations by promoting rapid protein accumulation, as the direct result of impairments in protein removal, which assumes protein synthesis remains relatively unchanged during proteasome inhibition. We conducted experimentation using established proteasome inhibitors and primary rat neuron cultures in order to elucidate whether proteasome inhibition had any effect on neuronal protein synthesis. Proteasome inhibition impaired neuronal protein synthesis, with concentrations of inhibitor necessary to significantly inhibit protein synthesis similar to the concentrations necessary to induce subsequent neuron death. The inhibition of protein synthesis was reversible during the first 6 h of treatment, with the neurotoxicity of proteasome inhibition reversible during the first 12 h of treatment. These studies are the first to demonstrate a potentially important interplay between the proteasome and protein synthesis in neurons, and the first to identify that some effects of proteasome inhibition are reversible in neurons. Together these findings have important implications for understanding proteasome inhibition as a potential contributor to aging and age-related disease.

Oxidative damage, protein synthesis, and protein degradation in Alzheimer's disease.

A large number of studies has firmly established that increases in oxidative damage occurs in Alzheimer's disease (AD). Such studies have demonstrated that increased in oxidative damage selectively occurs within the brain regions involved in regulating cognitive performance. Studies from our laboratory and others have provided experimental evidence that increased levels of oxidative damage occur in subjects with Mild Cognitive Impairment (MCI), which is believed to be one of the earliest stages of AD, and is a condition which is devoid of dementia or the extensive neurofibrillary pathology and neuritic plaque deposition observed in AD. Together, these data support a role for the accrual of oxidative damage potentially serving as an early event that then initiates the development of cognitive disturbances and pathological features observed in AD. Recent studies from our laboratory have demonstrated that a decline in protein synthesis capabilities occurs in the same brain regions which exhibit increased levels of oxidative damage in MCI and AD subjects. The focus of this review is to describe the large number of studies which suggest protein synthesis may be one of the earliest cellular processes disrupted by oxidative damage in AD. Taken together, these findings have important implications for understanding the molecular and cellular basis of AD, understanding the basis for oxidative stress in AD, and may have important implications for studies involving proteomics and proteolysis in AD.

Oxidative stress alters neuronal RNA- and protein-synthesis: Implications for neural viability.

Recent studies have demonstrated that impaired protein synthesis occurs in several neurodegenerative conditions associated with oxidative stress. Studies have also demonstrated that administration of oxidative stressors is sufficient to impair different and discrete regulatory aspects of protein synthesis in neural cells, with the majority of these studies focused on the effects of oxidative stressors towards initiation factors. Currently, little is known with regards to oxidative stress effects on the rates of RNA- and protein-synthesis, or the relationship between oxidant-induced impairments in RNA-/protein-synthesis to subsequent neuron death. In the present study, we demonstrate that administration of an oxidative stressor (hydrogen peroxide) induces a significant and time-dependent decrease in both RNA- and protein-synthesis in primary neurons and neural SH-SY5Y cells. Increases in RNA oxidation and disruption of ribosome complexes were selectively observed following the longer durations of oxidant exposure. Significant correlations between the loss of RNA- and protein-synthesis and the amount of oxidant-induced neuron death were also observed. Interestingly, the addition of a protein synthesis inhibitor (cycloheximide) did not significantly alter the amount of neuron death induced by the oxidative stressor. These data demonstrate that oxidant exposure promotes a time-dependent decrease in both RNA- and protein-synthesis in neurons, and demonstrate a role for elevations in RNA oxidation and ribosome dysfunction as potential mediators of impaired protein synthesis. These data also suggest that there is a complex relationship between the ability of oxidative stressors to modulate RNA- and protein-synthesis, and the ability of oxidative stressors to ultimately induce neuron death.

Oxidative inactivation of the proteasome in Alzheimer's disease.

In the present study we isolated proteasome complexes from control, mild cognitive impairment (MCI), and Alzheimer's disease (AD) subjects. No significant difference in the amount of proteasomes was detected across the different groups, although impairments in chymotrypsin-like proteasome activity was observed in AD subjects. Large impairments in proteasome- mediated degradation of an oxidized protein substrate was observed in MCI and AD subjects. Incubation with a reducing agent (DTT) had no significant effect on proteasome chymotrypsin-like activity, but fully restored proteasome-mediated protein degradation in MCI and AD subjects. Proteasomes from AD subjects exhibited elevations in protein carbonyls, 4-hydroxynonenal-conjugation, and neuroprostane-conjugation. Together, these data confirm that impairments in the function of purified proteasomes occurs in the earliest stages of AD, and directly support a role for oxidative inactivation contributing to declines in proteasome function in AD.

Ribosome dysfunction is an early event in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive and devastating disorder that is often preceded by mild cognitive impairment (MCI). In the present study, we report that in multiple cortical areas of MCI and AD subjects, there is a significant impairment in ribosome function that is not observed in the cerebellum of the same subjects. The impairment in ribosome function is associated with a decreased rate and capacity for protein synthesis, decreased ribosomal RNA and tRNA levels, and increased RNA oxidation. No alteration in the level of initiation factors was observed in the brain regions exhibiting impairments in protein synthesis. Together, these data indicate for the first time that impairments in protein synthesis may be one of the earliest neurochemical alterations in AD and directly demonstrate that the polyribosome complex is adversely affected early in the development of AD. These data have important implications for AD studies involving proteomics and studies analyzing proteolysis in AD, indicate that oxidative damage may contribute to decreased protein synthesis, and suggest a role for alterations in protein synthesis as a novel contributor to the onset and development of AD.

Evaluation of rage isoforms, ligands, and signaling in the brain.

Since the identification of the receptor for advanced glycosylation end products (RAGE) in 1992, there have been tremendous strides made in our understanding of the role RAGE receptors play in a variety of physiological and pathological processes. Despite such progress, several fundamental aspects of RAGE expression and RAGE function remain largely unanswered. In particular, while multiple forms of the RAGE receptor are known to exist, little is known with regards to how these different isoforms of the RAGE receptor work together to mediate RAGE signaling. For example, some forms of the RAGE receptor may promote deleterious feed-forward pathways, while others may serve to inhibit deleterious activation of the RAGE receptor. Additionally, important questions remain with regards to the intracellular domain of the full-length RAGE receptor, and the specifics surrounding how intracellular signaling pathways become activated via the RAGE family of receptors. The focus of this review is to address each of these important issues, as well as other key aspects of RAGE biology, and discuss how they are important for both our understanding of the physiological and pathological roles of RAGE signaling within the brain.

Proteasome regulation of oxidative stress in aging and age-related diseases of the CNS.

Proteasome-mediated protein degradation is responsible for a large percentage of bulk protein turnover, particularly the degradation of short-lived and oxidized proteins. Increasing evidence suggests that proteasome inhibition occurs during the aging of the central nervous system (CNS), and in a variety of age-related disorders of the CNS. The focus of this review is to discuss the role of the proteasome as a regulator of oxidative stress, with preservation of proteasome function playing an important role in preventing oxidative stress, and proteasome inhibition playing an important role as a mediator of oxidative stress. In particular, this review will describe experimental evidence that proteasome inhibition is sufficient to induce mitochondrial dysfunction, increase reactive oxygen species generation, elevate RNA and DNA oxidation, and promote protein oxidation. Taken together, these data indicate that the proteasome is an important regulator of oxidative damage in the CNS, and suggest that proteasome inhibition may serve as an important switch for the induction of oxidative stress in the CNS. Additionally we discuss the likelihood that the 20S proteasome and 26S proteasome may play different roles in regulating oxidative stress and neurotoxicity in the aging CNS, and in age-related disorders of the CNS.

LMP2 knock-out mice have reduced proteasome activities and increased levels of oxidatively damaged proteins.

The proteasome is a large intracellular protease, composed of multiple subunits, that is present in all eukaryotic cells. Proteasome inhibition is known to occur during normal aging, and is believed to contribute towards an age-related increase in oxidative stress, although at present the mechanisms responsible for mediating age-related changes in proteasome activity have not been elucidated. At present the relationship between proteasome subunit expression, proteasome activity, and protein oxidation during normal aging has not been elucidated. In the present study we observed that the absence of LMP2, a specific proteasome subunit, decreases proteasome activities in both the brain and liver, with increased levels of protein oxidation occurring in both tissues. Results from this study demonstrate for the first time that individual proteasome subunits are important for the regulation of age-related changes in both proteasome activity and protein oxidation.

RNA interference toward UMP1 induces proteasome inhibition in Saccharomyces cerevisiae: evidence for protein oxidation and autophagic cell death.

The proteasome is a large intracellular protease that is responsible for a large portion of intracellular proteolysis, in particular the degradation of a majority of short-lived and oxidized proteins. Inhibition of proteasome function occurs in response to multiple stressors, with proteasome inhibition sufficient for the induction of a wide range of cytotoxic processes. Although considerable advances have been made in the understanding of the proteasome, and the effects of proteasome inhibition, our understanding of these topics in Saccharomyces cerevisiae has been slowed by the inability of proteasome inhibitors to penetrate and/or be retained in S. cerevisiae. Expression of UMP1 is necessary for proteasome assembly in S. cerevisiae, and in the present study we examined the effectiveness of RNA interference for UMP1 as a means of achieving proteasome inhibition in S. cerevisiae. Induction of RNA interference for UMP1 resulted in a dramatic decrease in UMP1 at the protein level, which was not observed in cells transformed with control vector. RNA interference caused an impairment in proteasome function, and increase in protein oxidation, with proteins involved in both stress response and energy metabolism showing increased oxidation. Interestingly, RNA interference induced cell death that seemed to be autophagic in nature, suggesting possible cross talk between the proteasome and the autophagic proteolytic pathways. Taken together, these data indicate that RNA interference may be a useful model with which to study the effects of proteasome inhibition in S. cerevisiae and demonstrate the ability of proteasome inhibition to induce cytotoxic alterations in S. cerevisiae.

Modulation of apolipoprotein E and interleukin-1beta in the aging liver.

With aging there is a decline in liver function that includes reductions in hepatic blood flow, metabolite clearance, and impaired tissue repair following injury. An increase in inflammatory signaling is often observed during aging, with inflammation potentially mediating age-related changes in the liver. Apolipoprotein E (ApoE) is primarily produced by the liver and has been shown to possess anti-inflammatory properties in a variety of paradigms and experimental settings. In the present study, we correlated age-related alterations in ApoE expression with age-related changes in the cytokine interleukin-1 beta (IL-1beta), in the liver of rats of increasing age. A significant increase in ApoE mRNA, and a significant decrease in IL-1beta mRNA, was seen with increasing age. At the protein level, ApoE expression increased with aging, although IL-1beta expression remained constant. No correlation was found between ApoE and IL-1beta expression at the mRNA or protein level. Taken together, these studies demonstrate that ApoE expression is altered during normal aging, and indicates that there is no correlation between ApoE and IL-1beta expression in the aging liver.

Splice variants of the receptor for advanced glycosylation end products (RAGE) in human brain.

Previous studies indicate that the receptor for advanced glycosylation end products (RAGE) plays an important role in multiple pathological processes, including Alzheimer's disease. Currently there are three established isoforms of the RAGE receptor, with each isoform generated as the result of alternative splicing. It is presently unclear which of the RAGE isoforms are normally expressed in the human brain, nor has it been determined if additional RAGE isoforms exist in the human brain. In the present study we demonstrate for the first time that each of the three established RAGE isoforms, as well as three previously unidentified RAGE splicing variants, are normally expressed in the human brain. These data suggest that RAGE may have multiple functions in the human brain, mediated by the individual or coordinated efforts of the different RAGE isoforms, with alternative splicing generating individual RAGE isoforms that specifically interact with the various ligands present in the brain.

Lower expression level of two RAGE alternative splicing isoforms in Alzheimer's disease.

Alternative splicing (AS) is a common phenomenon in gene expression of eukaryotic organisms, especially in mammals, producing multiple gene isoforms from a single gene that involve in physiological and pathological processes. Receptor for advanced glycation end products (RAGE) has multiple AS isoforms with significant tissue and organism specificity. RAGE signaling has been reported involved in the onset and development of Alzheimer's disease (AD) and the roles of RAGE AS isoforms have not yet been fully illustrated in AD pathogenesis. In the present study, two of RAGE AS isoforms (RAGEΔ and sRAGEΔ) were investigated in the human brain specimens from AD and age-matched control subjects. The expression of these isoforms was found brain-region specific, and significant lower expression levels of both RAGEΔ and sRAGEΔ were detected in multiple brain regions of AD subjects than control subjects. Data indicated tight association between the AS isoforms (RAGEΔ and sRAGEΔ) and neurodegeneration. An antagonistic pairing model has been suggested to explain the working mechanism of AS isoforms on gene regulation and pathological development.

Autophagy, proteasomes, lipofuscin, and oxidative stress in the aging brain.

In order to successfully respond to stress all cells rely on the ability of the proteasomal and lysosomal proteolytic pathways to continually maintain protein turnover. Increasing evidence suggests that as part of normal aging there are age-related impairments in protein turnover by the proteasomal proteolytic pathway, and perturbations of the lysosomal proteolytic pathway. Furthermore, with numerous studies suggest an elevated level of a specialized form of lysosomal proteolysis (autophagy or macroautophagy) occurs during the aging of multiple cell types. Age-related alterations in proteolysis are believed to contribute to a wide variety of neuropathological manifestations including elevations in protein oxidation, protein aggregation, and cytotoxicity. Within the brain altered protein turnover is believed to contribute to elevations in multiple forms of protein aggregation ranging from tangle and Lewy body formation, to lipofuscin-ceroid accumulation. In this review we discuss and summarize evidence for proteolytic alterations occurring in the aging brain, the contribution of oxidative stress to disruption of protein turnover during normal aging, the evidence for cross-talk between the proteasome and lysosomal proteolytic pathways in the brain, and explore the contribution of altered proteolysis as a mediator of oxidative stress, neuropathology, and neurotoxicity in the aging brain.

The proteasome in brain aging.

The proteasome is a large intracellular protease, present in all cells of the central nervous system (CNS), that is responsible for the majority of intracellular protein degradation. In particular, the proteasome is responsible for the degradation of most oxidized, aggregated, and misfolded proteins. The importance of proteasome activity to neuronal homeostasis is highlighted by previous studies demonstrating that proteasome inhibition alone is sufficient to induce neuron death in vitro. Recent studies indicate that alterations in proteasome activity may occur during, and possibly contribute to, the aging process. These data raise the possibility that alterations in the proteasome proteolytic pathway may contribute to the elevations in protein oxidation, protein aggregation, and neurodegeneration evident in the aging CNS. The focus of this review is to describe what is currently known about the proteasome in the CNS, describe established age-related alterations in proteasome biology, and to discuss how such alterations in proteasome biology may ultimately contribute to the aging of the CNS.