The effect of botulinum toxin on ureteral inflammation.

PURPOSE: The impact of onabotulinum toxin type A (BoNT-A) on bladder afferent nerve pathways and chemosensory functions is an active area of investigation. There may be a role for BoNT-A in disorders of the ureter; however, no histologic studies have assessed the effects of BoNT-A on ureteral tissue. Our objective was to develop an animal model of ureteral inflammation and determine the impact of ureteral BoNT-A instillation on known mechanisms of inflammation. METHODS: The safety and feasibility of a novel animal model of ureteral inflammation was assessed. Through open cystotomy, the effect of ureteral BoNT-A instillation on inflammation was determined through H&E, masson's trichrome, Ki-67 stain, and prostaglandin E (PGE) synthase expression, a known marker of pain and inflammation in ureteral tissue. Urothelial microstructure was assessed using electron microscopy and standard histologic techniques. RESULTS: All experiments were carried to completion, and no systemic signs of botulinum toxicity were seen. BoNT-A exposure was associated with a decrease in PGE synthase expression in a dose-dependent fashion. BoNT-A exposure was not found to impact collagen deposition or cell proliferation. Disruption of tight junctions between urothelial cells was observed under conditions of inflammation. CONCLUSION: We describe the feasibility of a novel in vivo model of ureteral inflammation and report the first histologic study of the effects of BoNT-A on the ureter. Preliminary findings show that BoNT-A attenuates ureteral PGE synthase expression under conditions of inflammation. The application of BoNT-A may provide anti-inflammatory and analgesic effects in the context of ureteral disorders.

Slc6a3-dependent expression of a CAPS-associated Nlrp3 allele results in progressive behavioral abnormalities and neuroinflammation in aging mice.

BACKGROUND: An association between neuroinflammation and age-related neurologic disorders has been established but the molecular mechanisms and cell types involved have not been thoroughly characterized. Activity of the proinflammatory NLRP3 inflammasome is implicated in Alzheimer's and Parkinson's disease and our recent studies in patients suggest that dopaminergic neurons within the degenerating mesencephalon express NLRP3 throughout the progression of PD. Here, we directly test the impact of enhanced inflammasome activity in mesencephalic neurons by characterizing motor function, tissue integrity, and neuroinflammation in aging mice harboring hyperactivating mutations within the endogenous murine Nlrp3 locus, enabled only in cells expressing the dopaminergic neuron-specific Slc6a3 promoter. METHODS: We compared mice harboring inducible alleles encoding the cryopyrin-associated periodic syndrome activating mutations Nlrp3A350V and Nlrp3L351P inserted into the endogenous mouse Nlrp3 locus. Tissue specific expression was driven by breeding these animals with mice expressing Cre recombinase under the control of the dopaminergic neuron-specific Slc6a3 promoter. The experimental mice, designed to express hyperactive NLRP3 only when the endogenous mouse Nlrp3 promotor is active in dopaminergic neurons, were analyzed throughout 18 months of aging using longitudinal motor function assessments. Biochemical and histologic analyses of mesencephalic tissues were conducted in 1- and 18-month-old animals. RESULTS: We observed progressive and significant deficits in motor function in animals expressing Nlrp3L351P, compared with animals expressing Nlrp3WT and Nlrp3A350V. Age-dependent neuroinflammatory changes in the mesencephalon were noted in all animals. Analysis of GFAP-immunoreactive astrocytes in the substantia nigra revealed a significant increase in astrocyte number in animals expressing Nlrp3L351P compared with Nlrp3WT and Nlrp3A350V. Further analysis of Nlrp3L351P striatal tissues indicated genotype specific gliosis, elevated Il1b expression, and both morphologic and gene expression indicators of proinflammatory A1 astrocytes. CONCLUSIONS: Dopaminergic neurons have the potential to accumulate NLRP3 inflammasome activators with age, including reactive oxygen species, dopamine metabolites, and misfolded proteins. Results indicate the Nlrp3 locus is active in dopaminergic neurons in aging mice, and that the hyperactive Nlrp3L351P allele can drive neuroinflammatory changes in association with progressive behavioral deficits. Findings suggest neuronal NLRP3 inflammasome activity may contribute to neuroinflammation observed during normal aging and the progression of neurologic disorders.

NLRP3 inflammasome in neurodegenerative disease.

Neurodegenerative diseases are characterized by a dysregulated neuro-glial microenvironment, culminating in functional deficits resulting from neuronal cell death. Inflammation is a hallmark of the neurodegenerative microenvironment and despite a critical role in tissue homeostasis, increasing evidence suggests that chronic inflammatory insult can contribute to progressive neuronal loss. Inflammation has been studied in the context of neurodegenerative disorders for decades but few anti-inflammatory treatments have advanced to clinical use. This is likely due to the related challenges of predicting and mitigating off-target effects impacting the normal immune response while detecting inflammatory signatures that are specific to the progression of neurological disorders. Inflammasomes are pro-inflammatory cytosolic pattern recognition receptors functioning in the innate immune system. Compelling pre-clinical data has prompted an intense interest in the role of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in neurodegenerative disease. NLRP3 is typically inactive but can respond to sterile triggers commonly associated with neurodegenerative disorders including protein misfolding and aggregation, mitochondrial and oxidative stress, and exposure to disease-associated environmental toxicants. Clear evidence of enhanced NLRP3 inflammasome activity in common neurodegenerative diseases has coincided with rapid advancement of novel small molecule therapeutics making the NLRP3 inflammasome an attractive target for near-term interventional studies. In this review, we highlight evidence from model systems and patients indicating inflammasome activity in neurodegenerative disease associated with the NLRP3 inflammasome's ability to recognize pathologic forms of amyloid-ß, tau, and α-synuclein. We discuss inflammasome-driven pyroptotic processes highlighting the potential utility of evaluating extracellular inflammasome-related proteins in the context of biomarker discovery. We complete the report by pointing out gaps in our understanding of intracellular modifiers of inflammasome activity and mechanisms regulating the resolution of inflammasome activation. The literature review and perspectives provide a conceptual platform for continued analysis of inflammation in neurodegenerative diseases through the study of inflammasomes and pyroptosis, mechanisms of inflammation and cell death now recognized to function in multiple highly prevalent neurological disorders.

Exposomics as a tool to investigate differences in health and disease by sex and gender.

The health and disease of an individual is mediated by their genetics, a lifetime of environmental exposures, and interactions between the two. Genetic or biological sex, including chromosome composition and hormone expression, may influence both the types and frequency of environmental exposures an individual experiences, as well as the biological responses an individual has to those exposures. Gender identity, which can be associated with social behaviors such as expressions of self, may also mediate the types and frequency of exposures an individual experiences. Recent advances in exposome-level analysis have progressed our understanding of how environmental factors affect health outcomes; however, the relationship between environmental exposures and sex- and gender-specific health remains underexplored. The comprehensive, non-targeted, and unbiased nature of exposomic research provides a unique opportunity to systematically evaluate how environmental exposures interact with biological sex and gender identity to influence health. In this forward-looking narrative review, we provide examples of how biological sex and gender identity influence environmental exposures, discuss how environmental factors may interact with biological processes, and highlight how an intersectional approach to exposomics can provide critical insights for sex- and gender-specific health sciences.

Facile method to incorporate high-affinity ACAT/SOAT1 inhibitor F12511 into stealth liposome-based nanoparticle and demonstration of its efficacy in blocking cholesteryl ester biosynthesis without overt toxicity in neuronal cell culture.

BACKGROUND: Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors have been considered as potential therapeutic agents to treat several diseases, including Alzheimer's disease, atherosclerosis, and cancer. While many ACAT inhibitors are readily available, methods to encapsulate them as nanoparticles have not been reported. NEW METHOD: We report a simple method to encapsulate ACAT inhibitors, using the potent hydrophobic ACAT inhibitor F12511 as an example. By mixing DSPE-PEG2000, egg phosphatidylcholine (PC), and F12511 in ethanol, followed by drying, resuspension and sonication in buffer, we show that F12511 can be encapsulated as stealth liposomes at high concentration. RESULTS: We successfully incorporated F12511 into nanoparticles and found that increasing PC in the nanoparticles markedly increased the amount of F12511 incorporated in stealth liposomes. The nanoparticles containing F12511 (Nanoparticle F) exhibit average size of approximately 200 nm and are stable at 4 ºC for at least 6 months. Nanoparticle F is very effective at inhibiting ACAT in human and mouse neuronal and microglial cell lines. Toxicity tests using mouse primary neuronal cells show that F12511 alone or Nanoparticle F added at concentrations from 2 to 10 µM for 24-, 48-, and 72-hours produces minimal, if any, toxicity. COMPARISON WITH EXISTING METHOD(S): Unlike existing methods, the current method is simple, cost effective, and can be expanded to produce tagged liposomes to increase specificity of delivery. This also offers opportunity to embrace water soluble agent(s) within the aqueous compartment of the nanoparticles for potential combinatorial therapy. CONCLUSIONS: This method shows promise for delivery of hydrophobic ACAT inhibitors at high concentration in vivo.

Cross-species metabolomic analysis of tau- and DDT-related toxicity.

Exposure to the pesticide dichlorodiphenyltrichloroethane (DDT) has been associated with increased risk of Alzheimer's disease (AD), a disease also associated with hyperphosphorylated tau (p-tau) protein aggregation. We investigated whether exposure to DDT can exacerbate tau protein toxicity in Caenorhabditiselegans using a transgenic strain that expresses human tau protein prone to aggregation by measuring changes in size, swim behavior, respiration, lifespan, learning, and metabolism. In addition, we examined the association between cerebrospinal fluid (CSF) p-tau protein-as a marker of postmortem tau burden-and global metabolism in both a human population study and in C. elegans, using the same p-tau transgenic strain. From the human population study, plasma and CSF-derived metabolic features associated with p-tau levels were related to drug, amino acid, fatty acid, and mitochondrial metabolism pathways. A total of five metabolites overlapped between plasma and C. elegans, and four between CSF and C. elegans. DDT exacerbated the inhibitory effect of p-tau protein on growth and basal respiration. In the presence of p-tau protein, DDT induced more curling and was associated with reduced levels of amino acids but increased levels of uric acid and adenosylselenohomocysteine. Our findings in C. elegans indicate that DDT exposure and p-tau aggregation both inhibit mitochondrial function and DDT exposure can exacerbate the mitochondrial inhibitory effects of p-tau aggregation. Further, biological pathways associated with exposure to DDT and p-tau protein appear to be conserved between species.

Bbc3 Loss Enhances Survival and Protein Clearance in Neurons Exposed to the Organophosphate Pesticide Chlorpyrifos.

Exposure to environmental toxicants can increase the risk of developing age-related neurodegenerative disorders. Exposure to the widely used organophosphate pesticide chlorpyrifos (CPF) is associated with increased risk of developing Alzheimer's disease and Parkinson's disease, but the cellular mechanisms underlying CPF toxicity in neurons are not completely understood. We evaluated CPF toxicity in mouse primary cortical neuronal cultures, using RNA-sequencing to identify cellular pathways modulated by CPF. CPF exposure altered the expression of genes associated with intrinsic apoptosis, significantly elevating expression of the pro-apoptotic mediator Bbc3/Puma. Bbc3 loss attenuated CPF driven neurotoxicity, induction of other intrinsic apoptosis regulatory genes including Trp53 and Pmaip1 (encoding the NOXA protein), and cleavage of apoptosis executors caspase 3 and poly (ADP-ribose) polymerase (PARP). CPF exposure was associated with enhanced expression of endoplasmic reticulum stress-related genes and proteins and the accumulation of high molecular weight protein species in primary neuronal cultures. No evidence of alterations in the ubiquitin-proteosome system were observed, however, autophagy-related proteins were upregulated in CPF-treated Bbc3-/- neuronal cultures compared with identically exposed WT cultures. Elevated autophagy-related protein expression in Bbc3-/- neuronal cultures was associated with a reduction in CPF-induced high molecular weight alpha-synuclein and tau immunoreactive protein aggregates. Studies indicate that Bbc3-/- neuronal cultures enhance the endoplasmic reticulum stress response and upregulate protein clearance mechanisms as a component of resistance to CPF-mediated toxicity.

Lifestyle Factors and Parkinson's Disease Risk in a Rural New England Case-Control Study.

INTRODUCTION: Parkinson's disease (PD) is an age-related neurodegenerative disease likely caused by complex interactions between genetic and environmental risk factors. Exposure to pesticides, toxic metals, solvents, and history of traumatic brain injury have been implicated as environmental risk factors for PD, underscoring the importance of identifying risk factors associated with PD across different communities. METHODS: We conducted a questionnaire-based case-control study in a rural area on the New Hampshire/Vermont border, enrolling PD patients and age- and sex-matched controls from the general population between 2017 and 2020. We assessed frequent participation in a variety of recreational and occupational activities and surveyed potential chemical exposures. RESULTS: Suffering from "head trauma or a concussion" prior to diagnosis was associated with a fourfold increased risk of PD. Adjustment for head trauma negated any risk of participation in "strenuous athletic activities." We observed a 2.7-fold increased risk of PD associated with activities involving lead (adjusted p=0.038). CONCLUSION: Implicating these factors in PD risk favors public health efforts in exposure mitigation while also motivating future work mechanisms and intervention opportunities.

Plasma-borne indicators of inflammasome activity in Parkinson's disease patients.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor and non-motor symptoms and loss of dopaminergic neurons of the substantia nigra. Inflammation and cell death are recognized aspects of PD suggesting that strategies to monitor and modify these processes may improve the management of the disease. Inflammasomes are pro-inflammatory intracellular pattern recognition complexes that couple these processes. The NLRP3 inflammasome responds to sterile triggers to initiate pro-inflammatory processes characterized by maturation of inflammatory cytokines, cytoplasmic membrane pore formation, vesicular shedding, and if unresolved, pyroptotic cell death. Histologic analysis of tissues from PD patients and individuals with nigral cell loss but no diagnosis of PD identified elevated expression of inflammasome-related proteins and activation-related "speck" formation in degenerating mesencephalic tissues compared with controls. Based on previous reports of circulating inflammasome proteins in patients suffering from heritable syndromes caused by hyper-activation of the NLRP3 inflammasome, we evaluated PD patient plasma for evidence of inflammasome activity. Multiple circulating inflammasome proteins were detected almost exclusively in extracellular vesicles indicative of ongoing inflammasome activation and pyroptosis. Analysis of plasma obtained from a multi-center cohort identified elevated plasma-borne NLRP3 associated with PD status. Our findings are consistent with others indicating inflammasome activity in neurodegenerative disorders. Findings suggest mesencephalic inflammasome protein expression as a histopathologic marker of early-stage nigral degeneration and suggest plasma-borne inflammasome-related proteins as a potentially useful class of biomarkers for patient stratification and the detection and monitoring of inflammation in PD.

Inflammasomes: An Emerging Mechanism Translating Environmental Toxicant Exposure Into Neuroinflammation in Parkinson's Disease.

Evidence indicates that complex gene-environment interactions underlie the incidence and progression of Parkinson's disease (PD). Neuroinflammation is a well-characterized feature of PD widely believed to exacerbate the neurodegenerative process. Environmental toxicants associated with PD, such as pesticides and heavy metals, can cause cellular damage and stress potentially triggering an inflammatory response. Toxicant exposure can cause stress and damage to cells by impairing mitochondrial function, deregulating lysosomal function, and enhancing the spread of misfolded proteins. These stress-associated mechanisms produce sterile triggers such as reactive oxygen species (ROS) along with a variety of proteinaceous insults that are well documented in PD. These associations provide a compelling rationale for analysis of sterile inflammatory mechanisms that may link environmental exposure to neuroinflammation and PD progression. Intracellular inflammasomes are cytosolic assemblies of proteins that contain pattern recognition receptors, and a growing body of evidence implicates the association between inflammasome activation and neurodegenerative disease. Characterization of how inflammasomes may function in PD is a high priority because the majority of PD cases are sporadic, supporting the widely held belief that environmental exposure is a major factor in disease initiation and progression. Inflammasomes may represent a common mechanism that helps to explain the strong association between exposure and PD by mechanistically linking environmental toxicant-driven cellular stress with neuroinflammation and ultimately cell death.