Role of the Exposome in Neurodegenerative Disease: Recent Insights and Future Directions.

Neurodegenerative diseases are increasing in prevalence and place a significant burden on society. The causes are multifactorial and complex, and increasing evidence suggests a dynamic interplay between genes and the environment, emphasizing the importance of identifying and understanding the role of lifelong exposures, known as the exposome, on the nervous system. This review provides an overview of recent advances toward defining neurodegenerative disease exposomes, focusing on Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. We present the current state of the field based on emerging data, elaborate on key themes and potential mechanisms, and conclude with limitations and future directions. ANN NEUROL 2024;95:635-652.

Dietary interventions improve diabetic kidney disease, but not peripheral neuropathy, in a db/db mouse model of type 2 diabetes.

Patients with type 2 diabetes often develop the microvascular complications of diabetic kidney disease (DKD) and diabetic peripheral neuropathy (DPN), which decrease quality of life and increase mortality. Unfortunately, treatment options for DKD and DPN are limited. Lifestyle interventions, such as changes to diet, have been proposed as non-pharmacological treatment options for preventing or improving DKD and DPN. However, there are no reported studies simultaneously evaluating the therapeutic efficacy of varying dietary interventions in a type 2 diabetes mouse model of both DKD and DPN. Therefore, we compared the efficacy of a 12-week regimen of three dietary interventions, low carbohydrate, caloric restriction, and alternate day fasting, for preventing complications in a db/db type 2 diabetes mouse model by performing metabolic, DKD, and DPN phenotyping. All three dietary interventions promoted weight loss, ameliorated glycemic status, and improved DKD, but did not impact percent fat mass and DPN. Multiple regression analysis identified a negative correlation between fat mass and motor nerve conduction velocity. Collectively, our data indicate that these three dietary interventions improved weight and glycemic status and alleviated DKD but not DPN. Moreover, diets that decrease fat mass may be a promising non-pharmacological approach to improve DPN in type 2 diabetes given the negative correlation between fat mass and motor nerve conduction velocity.

Bioavailability and Pharmacokinetics of Endoxifen in Female Rats and Dogs: Evidence to Support the Use of Endoxifen to Overcome the Limitations of CYP2D6-Mediated Tamoxifen Metabolism.

Endoxifen (ENDX) is an active metabolite of tamoxifen (TAM), a drug commonly used for the treatment of estrogen receptor-positive breast cancer and metabolized by CYP2D6. Genetic or drug-induced reductions in CYP2D6 activity decrease plasma ENDX concentrations and TAM efficacy. It was proposed that direct oral administration of ENDX would circumvent the issues related to metabolic activation of TAM by CYP2D6 and increase patient response. Here, we characterized the pharmacokinetics and oral bioavailability of ENDX in female rats and dogs. Additionally, ENDX exposure was compared following equivalent doses of ENDX and TAM. ENDX exposure was 100-fold and 10-fold greater in rats and dogs, respectively, with ENDX administration compared with an equivalent dose of TAM. In single-dose administration studies, the terminal elimination half-life and plasma clearance values were 6.3 hours and 2.4 L/h per kg in rats given 2 mg/kg i.v. ENDX and 9.2 hours and 0.4 L/h/kg in dogs given 0.5 mg/kg i.v. ENDX, respectively. Plasma concentrations above 0.1 µM and 1 µM ENDX were achieved with 20-mg/kg and 200-mg/kg doses in rats, and concentrations above 1 µM and 10 µM were achieved with 15-mg/kg and 100-mg/kg doses in dogs. Oral absorption of ENDX was linear in rats and dogs, with bioavailability greater than 67% in rats and greater than 50% in dogs. In repeated-dose administration studies, ENDX peak plasma concentrations reached 9 µM in rats and 20 µM in dogs following four daily doses of 200 mg/kg or 30 mg/kg ENDX, respectively. The results indicate that ENDX has high oral bioavailability, and therapeutic concentrations were maintained after repeated dosing. Oral dosing of ENDX resulted in substantially higher ENDX concentrations than a similar dose of TAM. These data support the ongoing development of ENDX to overcome the limitations associated with CYP2D6-mediated metabolism of TAM in humans. SIGNIFICANCE STATEMENT: This study presents for the first time the pharmacokinetics and bioavailability of endoxifen and three key tamoxifen metabolites following repeated oral dosing in female rats and dogs. This study reports that endoxifen has high oral bioavailability, and therapeutic concentrations were maintained after repeated dosing. On the basis of these data, Z-endoxifen (Z-ENDX) was developed as a drug based upon the hypothesis that oral administration of Z-ENDX would overcome the limitations of CYP2D6 metabolism required for full metabolic activation of tamoxifen.

The Dopamine D2 Receptor Contributes to the Spheroid Formation Behavior of U87 Glioblastoma Cells.

BACKGROUND: Glioblastoma multiforme (GBM) is a common and lethal cancer of the central nervous system. This cancer is difficult to treat because most anticancer therapeutics do not readily penetrate into the brain due to the tight control at the cerebrovascular barrier. Numerous studies have suggested that dopamine D2 receptor (D2R) antagonists, such as first generation antipsychotics, may have anticancer efficacy in vivo and in vitro. The role of the D2R itself in the anticancer effects is unclear, but there is evidence suggesting that D2R activation promotes stem-like and spheroid forming behaviors in GBM. OBJECTIVES: We aimed to observe the role of the dopamine D2R and its modulators (at selective concentrations) in spheroid formation and stemness of GBM cell line, U87MG, to clarify the validity of the D2R as a therapeutic target for cancer therapy. METHODS: Spheroid formation assays and Western blotting of the glioblastoma cell line, U87MG, were used to observe responses to treatment with the D2R agonists sumanirole, ropinirole, and 4-propyl-9-hydroxynaphthoxazine (PHNO); and the D2R antagonists thioridazine, pimozide, haloperidol, and remoxipride. Extreme limiting dilution analysis was done to determine the impact of sumanirole and remoxipride treatment on sphere-forming cell frequency. Proliferation was also measured by crystal violet staining. Stable lentiviral transduction of DRD2 or shDRD2 was used to validate the role of the D2R in assay behaviors. RESULTS: D2R antagonists thioridazine, pimozide, haloperidol, and remoxipride decrease spheroid formation behaviors at a selective 100 nmol/L concentration, while D2R agonists PHNO, sumanirole, and ropinirole increase the formation of spheroids. Similarly, 100 nmol/L remoxipride decreased sphere-forming cell frequency. These results were recapitulated with genetic overexpression and knockdown of the D2R, and combination experiments indicate that the D2R is required for the effects of the pharmacological modulators. Furthermore, spheroid proliferation and invasive capacity increased under treatment with 100 nmol/L sumanirole and decreased under treatment with 100 nmol/L thioridazine. Expression levels of the stemness markers Nestin and Sox2, as well as those of differentiation marker glial fibrillary acidic protein, were not altered by 100 nmol/L thioridazine or sumanirole for 72 h or continuous treatment with these compounds for 7 days during a spheroid formation assay. CONCLUSIONS: Signaling activity of the dopamine D2R may be involved in the spheroid formation phenotype in the context of the U87MG cell line. However, this modulation may not be due to alterations in stemness marker expression, but due to other factors that may contribute to spheroid formation, such as cell-cell adhesion or EGFR signaling.

Transcriptomic profiling of sciatic nerves and dorsal root ganglia reveals site-specific effects of prediabetic neuropathy.

Peripheral neuropathy (PN) is a severe and frequent complication of obesity, prediabetes, and type 2 diabetes characterized by progressive distal-to-proximal peripheral nerve degeneration. However, a comprehensive understanding of the mechanisms underlying PN, and whether these mechanisms change during PN progression, is currently lacking. Here, gene expression data were obtained from distal (sciatic nerve; SCN) and proximal (dorsal root ganglia; DRG) injury sites of a high-fat diet (HFD)-induced mouse model of obesity/prediabetes at early and late disease stages. Self-organizing map and differentially expressed gene analyses followed by pathway enrichment analysis identified genes and pathways altered across disease stage and injury site. Pathways related to immune response, inflammation, and glucose and lipid metabolism were consistently dysregulated with HFD-induced PN, irrespective of injury site. However, regulation of oxidative stress was unique to the SCN while dysregulated Hippo and Notch signaling were only observed in the DRG. The role of the immune system and inflammation in disease progression was supported by an increase in the percentage of immune cells in the SCN with PN progression. Finally, when comparing these data to transcriptomic signatures from human patients with PN, we observed conserved pathways related to metabolic dysregulation across species, highlighting the translational relevance of our mouse data. Our findings demonstrate that PN is associated with distinct site-specific molecular re-programming in the peripheral nervous system, identifying novel, clinically relevant therapeutic targets.

Metal mixtures associate with higher amyotrophic lateral sclerosis risk and mortality independent of genetic risk and correlate to self-reported exposures: a case-control study.

Background: The pathogenesis of amyotrophic lateral sclerosis (ALS) involves both genetic and environmental factors. This study investigates associations between metal measures in plasma and urine, ALS risk and survival, and exposure sources. Methods: Participants with and without ALS from Michigan provided plasma and urine samples for metal measurement via inductively coupled plasma mass spectrometry. Odds and hazard ratios for each metal were computed using risk and survival models. Environmental risk scores (ERS) were created to evaluate the association between exposure mixtures and ALS risk and survival and exposure source. ALS (ALS-PGS) and metal (metal-PGS) polygenic risk scores were constructed from an independent genome-wide association study and relevant literature-selected SNPs. Results: Plasma and urine samples from 454 ALS and 294 control participants were analyzed. Elevated levels of individual metals, including copper, selenium, and zinc, significantly associated with ALS risk and survival. ERS representing metal mixtures strongly associated with ALS risk (plasma, OR=2.95, CI=2.38-3.62, p<0.001; urine, OR=3.10, CI=2.43-3.97, p<0.001) and poorer ALS survival (plasma, HR=1.42, CI=1.24-1.63, p<0.001; urine, HR=1.52, CI=1.31-1.76, p<0.001). Addition of the ALS-PGS or metal-PGS did not alter the significance of metals with ALS risk and survival. Occupations with high potential of metal exposure associated with elevated ERS. Additionally, occupational and non-occupational metal exposures associated with measured plasma and urine metals. Conclusion: Metals in plasma and urine associated with increased ALS risk and reduced survival, independent of genetic risk, and correlated with occupational and non-occupational metal exposures. These data underscore the significance of metal exposure in ALS risk and progression.

Development and validation of a liquid chromatography-mass spectrometry assay for quantification of Z- and E- isomers of endoxifen and its metabolites in plasma from women with estrogen receptor positive breast cancer.

The selective estrogen receptor modifier tamoxifen (TAM) is widely used for the treatment of women with estrogen receptor positive (ER+ ) breast cancer. Endoxifen (ENDX) is a potent, active metabolite of TAM and is important for TAM's clinical activity. While multiple papers have been published regarding TAM metabolism, few studies have examined or quantified the metabolism of ENDX. To quantify ENDX and its metabolites in patient plasma samples, we have developed and validated a rapid, sensitive, and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of the E- and Z-isomers of ENDX (0.5-500 ng/ml) and the ENDX metabolites norendoxifen (1-500 and 0.5-500 ng/ml E and Z, respectfully), ENDX catechol (3.075-307.5 and 1.92-192 ng/ml E and Z, respectfully), 4'-hydroxy ENDX (0.33-166.5 and 0.33-333.5 ng/ml E and Z, respectfully), ENDX methoxycatechol (0.3-300 and 0.2-200 ng/ml E and Z, respectfully), and ENDX glucuronide (2-200 and 3-300 ng/ml E and Z, respectfully) in human plasma. Chromatographic separation was accomplished on a HSS T3 precolumn attached to an Poroshell 120 EC-C18 analytical column using 0.1 % formic acid/water and 0.1 % formic acid/methanol as eluents followed by MS/MS detection. The analytical run time was 6.5 min. Standard curves were linear (R2 ≥ 0.98) over the concentration ranges. The intra- and inter-day precision and accuracy, determined at high-, middle-, and low-quality control concentrations for all analytes, were within the acceptable range of 85 % and 115 %. The average percent recoveries were all above 90 %. The method was successfully applied to clinical plasma samples from a Phase I study of daily oral Z-ENDX.

Modeling the innate inflammatory cGAS/STING pathway: sexually dimorphic effects on microglia and cognition in obesity and prediabetes.

Introduction: The prevalence of obesity, prediabetes, and diabetes continues to grow worldwide. These metabolic dysfunctions predispose individuals to neurodegenerative diseases and cognitive impairment, including dementias such as Alzheimer's disease and Alzheimer's disease related dementias (AD/ADRD). The innate inflammatory cGAS/STING pathway plays a pivotal role in metabolic dysfunction and is an emerging target of interest in multiple neurodegenerative diseases, including AD/ADRD. Therefore, our goal was to establish a murine model to specifically target the cGAS/STING pathway to study obesity- and prediabetes-induced cognitive impairment. Methods: We performed two pilot studies in cGAS knockout (cGAS-/-) male and female mice designed to characterize basic metabolic and inflammatory phenotypes and examine the impact of high-fat diet (HFD) on metabolic, inflammatory, and cognitive parameters. Results: cGAS-/- mice displayed normal metabolic profiles and retained the ability to respond to inflammatory stimuli, as indicated by an increase in plasma inflammatory cytokine production in response to lipopolysaccharide injection. HFD feeding caused expected increases in body weight and decreases in glucose tolerance, although onset was accelerated in females versus males. While HFD did not increase plasma or hippocampal inflammatory cytokine production, it did alter microglial morphology to a state indicative of activation, particularly in female cGAS-/- mice. However, HFD negatively impacted cognitive outcomes in male, but not female animals. Discussion: Collectively, these results suggest that cGAS-/- mice display sexually dimorphic responses to HFD, possibly based on differences in microglial morphology and cognition.

Transcriptomic analysis of diabetic kidney disease and neuropathy in mouse models of type 1 and type 2 diabetes.

Diabetic kidney disease (DKD) and diabetic peripheral neuropathy (DPN) are common complications of type 1 (T1D) and type 2 (T2D) diabetes. However, the mechanisms underlying pathogenesis of these complications are unclear. In this study, we optimized a streptozotocin-induced db/+ murine model of T1D and compared it to our established db/db T2D mouse model of the same C57BLKS/J background. Glomeruli and sciatic nerve transcriptomic data from T1D and T2D mice were analyzed by self-organizing map and differential gene expression analysis. Consistent with prior literature, pathways related to immune function and inflammation were dysregulated in both complications in T1D and T2D mice. Gene-level analysis identified a high degree of concordance in shared differentially expressed genes (DEGs) in both complications and across diabetes type when using mice from the same cohort and genetic background. As we have previously shown a low concordance of shared DEGs in DPN when using mice from different cohorts and genetic backgrounds, this suggests that genetic background may influence diabetic complications. Collectively, these findings support the role of inflammation and indicate that genetic background is important in complications of both T1D and T2D.

Actin Up: An Overview of the Rac GEF Dock1/Dock180 and Its Role in Cytoskeleton Rearrangement.

Dock1, originally Dock180, was the first identified member of the Dock family of GTPase Exchange Factors. Early biochemical and genetic studies of Dock180 elucidated the functions and regulation of Dock180 and informed our understanding of all Dock family members. Dock180 activates Rac to stimulate actin polymerization in response to signals initiated by a variety of receptors. Dock180 dependent Rac activation is essential for processes such as apoptotic cell engulfment, myoblast fusion, and cell migration during development and homeostasis. Inappropriate Dock180 activity has been implicated in cancer invasion and metastasis and in the uptake of bacterial pathogens. Here, we give an overview of the history and current understanding of the activity, regulation, and impacts of Dock180.

Mouse pharmacokinetics and metabolism of the phenylurea thiocarbamate NSC 161128.

PURPOSE: NSC 161128, a phenylurea thiocarbamate, displays activity against the NCI60 anti-cancer cell line panel and xenograft models. The metabolite N-methyl-N'-phenylurea (M10) has been detected in animal plasma; however, detection and quantification of other putative NSC 161128 metabolites have not been undertaken. The purpose of this study was to characterize the pharmacokinetics and metabolism of NSC 161128 in mice and under in vitro conditions. METHODS: An LC-MS/MS assay was developed to evaluate stability and in vitro metabolism of NSC 161128 in liver microsomes and S9 fractions. Single-dose pharmacokinetic profiles for NSC 161128 and its metabolite M10 were obtained following intraperitoneal (I.P.) administration. RESULTS: A sensitive and specific positive ionization LC-MS/MS method for measuring NSC 161128 and its metabolites was developed. HPLC separation was achieved under gradient elution using an aqueous methanol mobile phase containing 0.05% formic acid and 0.05% ammonium hydroxide. The assay was linear over the range 1.0-1000 ng/mL. NSC 161128 was stable in aqueous solution and tissue culture media, but not in plasma, where rapid degradation of NSC 161128 to the metabolite M10 was observed. Following I.P. administration of a 200 mg/kg dose to male CD1 mice, the peak plasma concentration of NSC 161128 was 255 ng/mL after 5 min with a plasma half-life of 138 min. Potential bioactivation of NSC 161128 was explored using mouse S9. CONCLUSIONS: An analytical LC-MS/MS method was successfully developed for the detection and quantification of NSC 161128 and its metabolites. These results increase the understanding of NSC 161128 pharmacokinetic and metabolic properties.

Population Pharmacokinetics of Z-Endoxifen in Patients With Advanced Solid Tumors.

The purpose of this study was to develop and validate a population pharmacokinetic model for Z-endoxifen in patients with advanced solid tumors and to identify clinical variables that influence pharmacokinetic parameters. Z-endoxifen-HCl was administered orally once a day on a 28-day cycle (±3 days) over 11 dose levels ranging from 20 to 360 mg. A total of 1256 Z-endoxifen plasma concentration samples from 80 patients were analyzed using nonlinear mixed-effects modeling to develop a population pharmacokinetic model for Z-endoxifen. A 2-compartment model with oral depot and linear elimination adequately described the data. The estimated apparent total clearance, apparent central volume of distribution, and apparent peripheral volume of distribution were 4.89 L/h, 323 L, and 39.7 L, respectively, with weight-effect exponents of 0.75, 1, and 1, respectively. This model was used to explore the effects of clinical and demographic variables on Z-endoxifen pharmacokinetics. Weight, race on clearance, and aspartate aminotransferase on the absorption rate constant were identified as significant covariates in the final model. This novel population pharmacokinetic model provides insight regarding factors that may affect the pharmacokinetics of Z-endoxifen and may assist in the design of future clinical trials.

Therapeutic Potential of 5'-Methylschweinfurthin G in Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare but aggressive form of skin cancer predominantly caused by the human Merkel cell polyomavirus (MCPyV). Treatment for MCC includes excision and radiotherapy of local disease, and chemotherapy or immunotherapy for metastatic disease. The schweinfurthin family of natural compounds previously displayed potent and selective growth inhibitory activity against the NCI-60 panel of human-derived cancer cell lines. Here, we investigated the impact of schweinfurthin on human MCC cell lines. Treatment with the schweinfurthin analog, 5'-methylschweinfurth G (MeSG also known as TTI-3114), impaired metabolic activity through induction of an apoptotic pathway. MeSG also selectively inhibited PI3K/AKT and MAPK/ERK pathways in the MCPyV-positive MCC cell line, MS-1. Interestingly, expression of the MCPyV small T (sT) oncogene selectively sensitizes mouse embryonic fibroblasts to MeSG. These results suggest that the schweinfurthin family of compounds display promising potential as a novel therapeutic option for virus-induced MCCs.

Schweinfurthin natural products induce regression of murine melanoma and pair with anti-PD-1 therapy to facilitate durable tumor immunity.

Metastatic melanoma is a significant clinical problem with a 5-year survival rate of only 15-20%. Recent approval of new immunotherapies and targeted inhibitors have provided much needed options for these patients, in some cases promoting dramatic disease regressions. In particular, antibody-based therapies that block the PD-1/PD-L1 checkpoint inhibitory pathway have achieved an increased overall response rate in metastatic melanoma, yet durable response rates are reported only around 15%. To improve the overall and durable response rates for advanced-stage melanoma, combined targeted and immune-based therapies are under investigation. Here, we investigated how the natural products called schweinfurthins, which have selective anti-proliferative activity against many cancer types, impact anti-(α)PD-1-mediated immunotherapy of murine melanomas. Two different compounds efficiently reduced the growth of human and murine melanoma cells in vitro and induced plasma membrane surface localization of the ER-resident protein calreticulin in B16.F10 melanoma cells, an indicator of immunogenic cell death. In addition, both compounds improved αPD-1-mediated immunotherapy of established tumors in immunocompetent C57BL/6 mice either by delaying tumor progression or resulting in complete tumor regression. Improved immunotherapy was accomplished following only a 5-day course of schweinfurthin, which was associated with initial tumor regression even in the absence of αPD-1. Schweinfurthin-induced tumor regression required an intact immune system as tumors were unaffected in NOD scid gamma (NSG) mice. These results indicate that schweinfurthins improve αPD-1 therapy, leading to enhanced and durable anti-tumor immunity and support the translation of this novel approach to further improve response rates for metastatic melanoma.

ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation.

Hepatocyte growth factor (HGF) is a potent signaling factor that acts on epithelial cells, causing them to dissociate and scatter. This migration is coordinated by a number of small GTPases, such as ARF6 and Rac1. Active ARF6 is required for HGF-stimulated migration and intracellular levels of ARF6-GTP and Rac1-GTP increase following HGF treatment. During migration, cross talk between ARF6 and Rac1 occurs through formation of a multi-protein complex containing the ARF-GEF cytohesin-2, the scaffolding protein GRASP/Tamalin, and the Rac1-GEF Dock180. Previously, the role of ARF6 in this process was unclear. We have now found that ARF6 and ARF1 regulate trafficking of GRASP and Dock180 to the plasma membrane following HGF treatment. Trafficking of GRASP and Dock180 is impaired by blocking ARF6-mediated recycling pathways and is required for HGF-stimulated Rac1 activation. Finally, HGF treatment stimulates association of GRASP and Dock180. Inhibition of ARF6 trafficking pathways traps GRASP and Dock180 as a complex in the cell.

Schweinfurthins: Lipid Modulators with Promising Anticancer Activity.

The schweinfurthin family of compounds displays exciting potent and differential cytotoxicity against human cancer cell lines. Currently, the effect of schweinfurthins on tumor development and progression is being explored in animal models of cancer with promising results. The first schweinfurthin family member, vedelianin, was isolated in 1992, followed by other schweinfurthins in 1998. This opened up the door for the synthesis of additional analogs. At present, the focus of research lies on delineating the mechanism of schweinfurthin action and identifying the nature of sensitivity. It appears that many of the intracellular effects of schweinfurthins are due to, or impacted by, the effect of schweinfurthins on lipid metabolism, synthesis, and homeostasis. These effects include impaired trafficking from the trans-golgi network, disruption of lipid rafts, changes in oxysterol-binding protein activity, and interference with the isoprenoid biosynthesis pathway (IBP). Cancer cells are known to rely heavily on fatty acid, lipid, and sterol synthesis for growth and proliferation. Therefore, compounds that target these needs, such as schweinfurthins, display promise as novel therapeutics. This timely review will take an in-depth look at the history of schweinfurthins, their synthesis, where the research presently stands, and the questions that remain.