HESI workshop summary: Interpretation of developmental and reproductive toxicity endpoints and the impact on data interpretation of adverse events.

The Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (HESI-DART) group held a hybrid in-person and virtual workshop in Washington, DC, in 2022. The workshop was entitled, "Interpretation of DART in Regulatory Contexts and Frameworks." There were 154 participants (37 in person and 117 virtual) across 9 countries. The purpose of the workshop was to capture key consensus approaches used to assess DART risks associated with chemical product exposure when a nonclinical finding is identified. The decision-making process for determining whether a DART endpoint is considered adverse is critical because the outcome may have downstream implications (e.g., increased animal usage, modifications to reproductive classification and pregnancy labeling, impact on enrollment in clinical trials and value chains). The workshop included a series of webinar modules to train and engage in discussions with federal and international regulators, clinicians, academic investigators, nongovernmental organizations, contract research organization scientists, and private sector scientists on the best practices and principles of interpreting DART and new approach methodologies in the context of regulatory requirements and processes. Despite the differences in regulatory frameworks between the chemical and pharmaceutical sectors, the same foundational principles for data interpretation should be applied. The discussions led to the categorization of principles, which offer guidance for the systematic interpretation of data. Step 1 entails identifying any hazard by closely analyzing the data at the study endpoint level, while Step 2 involves assessing risk using weight of evidence. These guiding principles were derived from the collective outcomes of the workshop deliberations.

The Rehabilitation Potential of Neurostimulation for Mild Traumatic Brain Injury in Animal and Human Studies.

Neurostimulation carries high therapeutic potential, accompanied by an excellent safety profile. In this review, we argue that an arena in which these tools could provide breakthrough benefits is traumatic brain injury (TBI). TBI is a major health problem worldwide, with the majority of cases identified as mild TBI (mTBI). MTBI is of concern because it is a modifiable risk factor for dementia. A major challenge in studying mTBI is its inherent heterogeneity across a large feature space (e.g., etiology, age of injury, sex, treatment, initial health status, etc.). Parallel lines of research in human and rodent mTBI can be collated to take advantage of the full suite of neuroscience tools, from neuroimaging (electroencephalography: EEG; functional magnetic resonance imaging: fMRI; diffusion tensor imaging: DTI) to biochemical assays. Despite these attractive components and the need for effective treatments, there are at least two major challenges to implementation. First, there is insufficient understanding of how neurostimulation alters neural mechanisms. Second, there is insufficient understanding of how mTBI alters neural function. The goal of this review is to assemble interrelated but disparate areas of research to identify important gaps in knowledge impeding the implementation of neurostimulation.

Hindbrain Stimulation Modulates Object Recognition Discrimination Efficiency and Hippocampal Synaptic Connections.

(1) Background: The cerebellum is well known to have functionalities beyond the control of motor function. However, brain stimulation studies have not explored the potential of this region to impact downstream processes which are imperative to multiple neurological conditions. Our study aimed to look at preliminary evidence that hindbrain-targeted repetitive transcranial magnetic stimulation (rTMS) in mice could alter motor, cognitive and anxiety measures; (2) Methods: Male B6129SF2/J mice (n = 16) were given rTMS (n = 9) over lambda at 10 Hz for 10 min or Sham (n = 7) for 14 consecutive days. Mice then underwent a battery of behavioral measures. (3) Results: In the object recognition test, only rTMS-treated mice distinguished between the novel object at 5 min, whereas those that received Sham treatment continued to improve discrimination from 5 to 10 min. Additionally, over the 10 min test phase, rTMS-stimulated mice explored the objects less than the Sham mice. This was accompanied by increased colocalization of presynaptic and postsynaptic markers in the hippocampus in the rTMS mice (4) Conclusions: Hindbrain rTMS stimulation elicits improved processing speed in the object recognition test via structural plasticity mechanisms in the hippocampus and could provide additional ways of targeting these important substructures of the brain.

BDNF rs6265 Met carriers with alcohol use disorder show greater age-related decline of N-acetylaspartate in left dorsolateral prefrontal cortex.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is implicated in neuronal and glial cell growth and differentiation, synaptic plasticity, and apoptotic mechanisms. A single-nucleotide polymorphism of the BDNF rs6265 gene may contribute to the pattern and magnitude of brain metabolite abnormalities apparent in those with an Alcohol Use Disorder (AUD). We predicted that methionine (Met) carriers would demonstrate lower magnetic resonance spectroscopy (MRS) measures of N-acetylaspartate level (NAA) and greater age-related decline in NAA than valine (Val) homozygotes. METHODS: Veterans with AUD (n=95; 46±12 years of age, min = 25, max = 71) were recruited from VA Palo Alto residential treatment centers. Single voxel MRS, at 3 Tesla, was used to obtain NAA, choline (Cho) and creatine (Cr) containing compounds from the left dorsolateral prefrontal cortex (DLPFC). Metabolite spectra were fit with LC Model and NAA and Cho were standardized to total Cr level and NAA was also standardized to Cho. RESULTS: Val/Met (n=35) showed markedly greater age-related decline in left DLPFC NAA/Cr level than Val/Val (n=60); no differences in mean metabolite levels were observed between Val/Met and Val/Val. Val/Met demonstrated greater frequency of history of MDD and higher frequency of cannabis use disorder over 12 months prior to study. CONCLUSIONS: The greater age-related decline in left DLPFC NAA/Cr and the higher frequency of MDD history and Cannabis Use disorder in BDNF rs6265 Met carriers with AUD are novel and may have implications for non-invasive brain stimulation targeting the left DLFPC and other psychosocial interventions typically utilized in the treatment of AUD.

Costs and Benefits of Popular P-Value Correction Methods in Three Models of Quantitative Omic Experiments.

The multiple hypothesis testing problem is inherent in large-scale quantitative "omic" experiments such as mass spectrometry-based proteomics. Yet, tools for comparing the costs and benefits of different p-value correction methods under different experimental conditions are lacking. We performed thousands of simulations of omic experiments under a range of experimental conditions and applied correction using the Benjamini-Hochberg (BH), Bonferroni, and permutation-based false discovery proportion (FDP) estimation methods. The tremendous false discovery rate (FDR) benefit of correction was confirmed in a range of different contexts. No correction method can guarantee a low FDP in a single experiment, but the probability of a high FDP is small when a high number and proportion of corrected p-values are significant. On average, correction decreased sensitivity, but the sensitivity costs of BH and permutation were generally modest compared to the FDR benefits. In a given experiment, observed sensitivity was always maintained or decreased by BH and Bonferroni, whereas it was often increased by permutation. Overall, permutation had better FDR and sensitivity than BH. We show how increasing sample size, decreasing variability, or increasing effect size can enable the detection of all true changes while still correcting p-values, and we present basic guidelines for omic experimental design. Analysis of an experimental proteomic data set with defined changes corroborated these trends. We developed an R Shiny web application for further exploration and visualization of these models, which we call the Simulator of P-value Multiple Hypothesis Correction (SIMPLYCORRECT) and a high-performance R package, permFDP, for easy use of the permutation-based FDP estimation method.

A human brain vascular atlas reveals diverse mediators of Alzheimer's risk.

The human brain vasculature is of great medical importance: its dysfunction causes disability and death1, and the specialized structure it forms-the blood-brain barrier-impedes the treatment of nearly all brain disorders2,3. Yet so far, we have no molecular map of the human brain vasculature. Here we develop vessel isolation and nuclei extraction for sequencing (VINE-seq) to profile the major vascular and perivascular cell types of the human brain through 143,793 single-nucleus transcriptomes from 25 hippocampus and cortex samples of 9 individuals with Alzheimer's disease and 8 individuals with no cognitive impairment. We identify brain-region- and species-enriched genes and pathways. We reveal molecular principles of human arteriovenous organization, recapitulating a gradual endothelial and punctuated mural cell continuum. We discover two subtypes of human pericytes, marked by solute transport and extracellular matrix (ECM) organization; and define perivascular versus meningeal fibroblast specialization. In Alzheimer's disease, we observe selective vulnerability of ECM-maintaining pericytes and gene expression patterns that implicate dysregulated blood flow. With an expanded survey of brain cell types, we find that 30 of the top 45 genes that have been linked to Alzheimer's disease risk by genome-wide association studies (GWASs) are expressed in the human brain vasculature, and we confirm this by immunostaining. Vascular GWAS genes map to endothelial protein transport, adaptive immune and ECM pathways. Many are microglia-specific in mice, suggesting a partial evolutionary transfer of Alzheimer's disease risk. Our work uncovers the molecular basis of the human brain vasculature, which will inform our understanding of overall brain health, disease and therapy.

Repetitive Transcranial Magnetic Stimulation Improves Brain-Derived Neurotrophic Factor and Cholinergic Signaling in the 3xTgAD Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a debilitating disorder involving the loss of plasticity and cholinergic neurons in the cortex. Pharmaceutical treatments are limited in their efficacy, but brain stimulation is emerging as a treatment for diseases of cognition. More research is needed to determine the biochemical mechanisms and treatment efficacy of this technique. OBJECTIVE: We aimed to determine if forebrain repetitive transcranial magnetic stimulation can improve cortical BDNF gene expression and cholinergic signaling in the 3xTgAD mouse model of AD. METHODS: Both B6 wild type mice and 3xTgAD mice aged 12 months were given daily treatment sessions for 14 days or twice weekly for 6 weeks. Following treatment, brain tissue was extracted for immunological stains for plaque load, as well as biochemical analysis for BDNF gene expression and cholinergic signaling via acetylcholinesterase and choline acetyltransferase ELISA assays. RESULTS: For the 3xTgAD mice, both 14 days and 6 weeks treatment regimens resulted in an increase in BDNF gene expression relative to sham treatment, with a larger increase in the 6-week group. Acetylcholinesterase activity also increased for both treatments in 3xTgAD mice. The B6 mice only had an increase in BDNF gene expression for the 6-week group. CONCLUSION: Brain stimulation is a possible non-invasive and nonpharmaceutical treatment option for AD as it improves both plasticity markers and cholinergic signaling in an AD mouse model.

Improved object recognition memory using post-encoding repetitive transcranial magnetic stimulation.

BACKGROUND: Brain stimulation is known to affect canonical pathways and proteins involved in memory. However, there are conflicting results on the ability of brain stimulation to improve to memory, which may be due to variations in timing of stimulation. HYPOTHESIS: We hypothesized that repetitive transcranial magnetic stimulation (rTMS) given following a learning task and within the time period before retrieval could help improve memory. METHODS: We implanted male B6129SF2/J mice (n = 32) with a cranial attachment to secure the rTMS coil so that the mice could be given consistent stimulation to the frontal area whilst freely moving. Mice then underwent the object recognition test sampling phase and given treatment +3, +24, +48 h following the test. Treatment consisted of 10 min 10 Hz rTMS stimulation (TMS, n = 10), sham treatment (SHAM, n = 11) or a control group which did not do the behavior test or receive rTMS (CONTROL n = 11). At +72 h mice were tested for their exploration of the novel vs familiar object. RESULTS: At 72-h's, only the mice which received rTMS had greater exploration of the novel object than the familiar object. We further show that promoting synaptic GluR2 and maintaining synaptic connections in the perirhinal cortex and hippocampal CA1 are important for this effect. In addition, we found evidence that these changes were linked to CAMKII and CREB pathways in hippocampal neurons. CONCLUSION: By linking the known biological effects of rTMS to memory pathways we provide evidence that rTMS is effective in improving memory when given during the consolidation and maintenance phases.

Repetitive Transcranial Magnetic Stimulation as a Treatment for Veterans with Cognitive Impairment and Multiple Comorbidities.

BACKGROUND: Despite decades of research efforts, current treatments for Alzheimer's disease (AD) are of limited effectiveness and do not halt the progression of the disease and associated cognitive decline. Studies have shown that repetitive transcranial magnetic stimulation (rTMS) may improve cognition. OBJECTIVE: We conducted a pilot study to investigate the effect of rTMS on cognitive function in Veterans with numerous medical comorbidities. METHODS: Participants underwent 20 sessions, over the course of approximately 4 weeks, of 10 Hz rTMS at the left dorsolateral prefrontal cortex with intensity of 120% resting motor threshold. Outcome measures including memory, language, verbal fluency, and executive functions were acquired at baseline, end of treatment, and 4 months after the last rTMS session. Twenty-six Veterans completed the study (13 in the active rTMS group, 13 in the sham rTMS group). RESULTS: The study protocol was well-tolerated. Active, compared to sham, rTMS showed improved auditory-verbal memory at the end of treatment and at 4-month follow-up. However, the active rTMS group demonstrated a trend in decreased semantic verbal fluency at the end of treatment and at 4-month follow up. CONCLUSION: These preliminary results show rTMS is safe in general in this elderly Veteran population with multiple co-morbidities. Patients in the sham group showed an expected, slight decline in the California Verbal Learning Test scores over the course of the study, whereas the active treatment group showed a slight improvement at the 4-month post-treatment follow up. These effects need to be confirmed by studies of larger sample sizes.

Concordance of 3 alternative teratogenicity assays with results from corresponding in vivo embryo-fetal development studies: Final report from the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) DruSafe working group 2.

An IQ DruSafe working group evaluated the concordance of 3 alternative teratogenicity assays (rat whole embryo culture, rWEC; zebrafish embryo culture, ZEC; and murine embryonic stem cells, mESC) with findings from rat or rabbit embryo-fetal development (EFD) studies. Data for 90 individual compounds from 9 companies were entered into a database. In vivo findings were deemed positive if malformations or embryo-fetal lethality were reported in either species. Each company used their own criteria for deciding whether the alternative assay predicted the in vivo findings. Standard concordance parameters were calculated, positive and negative predictive values (PPV and NPV) were adjusted for the aggregate portfolio prevalence of positive compounds (established by a survey of participating companies), and positive and negative likelihood ratios (LR+ and iLR-) were calculated. Of the 3 assays, only rWEC data were robustly predictive, particularly for negative predictions (NPVadj = 92%). However, both LR+ (4.92) and iLR- (4.72) were statistically significant for the rWEC assay. When analyzed separately for rats, the NPVadj and iLR-values for the rWEC assay increased to 96% and 9.75, respectively. These data suggest that a negative rWEC outcome could defer or replace a rat EFD study in certain regulatory settings.

Development of a CNS-permeable reactivator for nerve agent exposure: an iterative, multi-disciplinary approach.

Nerve agents have experienced a resurgence in recent times with their use against civilian targets during the attacks in Syria (2012), the poisoning of Sergei and Yulia Skripal in the United Kingdom (2018) and Alexei Navalny in Russia (2020), strongly renewing the importance of antidote development against these lethal substances. The current standard treatment against their effects relies on the use of small molecule-based oximes that can efficiently restore acetylcholinesterase (AChE) activity. Despite their efficacy in reactivating AChE, the action of drugs like 2-pralidoxime (2-PAM) is primarily limited to the peripheral nervous system (PNS) and, thus, provides no significant protection to the central nervous system (CNS). This lack of action in the CNS stems from their ionic nature that, on one end makes them very powerful reactivators and on the other renders them ineffective at crossing the Blood Brain Barrier (BBB) to reach the CNS. In this report, we describe the use of an iterative approach composed of parallel chemical and in silico syntheses, computational modeling, and a battery of detailed in vitro and in vivo assays that resulted in the identification of a promising, novel CNS-permeable oxime reactivator. Additional experiments to determine acute and chronic toxicity are ongoing.

Dysregulation of brain and choroid plexus cell types in severe COVID-19.

Although SARS-CoV-2 primarily targets the respiratory system, patients with and survivors of COVID-19 can suffer neurological symptoms1-3. However, an unbiased understanding of the cellular and molecular processes that are affected in the brains of patients with COVID-19 is missing. Here we profile 65,309 single-nucleus transcriptomes from 30 frontal cortex and choroid plexus samples across 14 control individuals (including 1 patient with terminal influenza) and 8 patients with COVID-19. Although our systematic analysis yields no molecular traces of SARS-CoV-2 in the brain, we observe broad cellular perturbations indicating that barrier cells of the choroid plexus sense and relay peripheral inflammation into the brain and show that peripheral T cells infiltrate the parenchyma. We discover microglia and astrocyte subpopulations associated with COVID-19 that share features with pathological cell states that have previously been reported in human neurodegenerative disease4-6. Synaptic signalling of upper-layer excitatory neurons-which are evolutionarily expanded in humans7 and linked to cognitive function8-is preferentially affected in COVID-19. Across cell types, perturbations associated with COVID-19 overlap with those found in chronic brain disorders and reside in genetic variants associated with cognition, schizophrenia and depression. Our findings and public dataset provide a molecular framework to understand current observations of COVID-19-related neurological disease, and any such disease that may emerge at a later date.

Moving back in the brain to drive the field forward: Targeting neurostimulation to different brain regions in animal models of depression and neurodegeneration.

BACKGROUND: Repetitive transcranial magnetic stimulation is a promising noninvasive therapeutic tool for a variety of brain-related disorders. However, most therapeutic protocols target the anterior regions, leaving many other areas unexplored. There is a substantial therapeutic potential for stimulating various brain regions, which can be optimized in animal models. NEW METHOD: We illustrate a method that can be utilized reliably to stimulate the anterior or posterior brain in freely moving rodents. A coil support device is surgically attached onto the skull, which is used for consistent coil placement over the course of up to several weeks of stimulation sessions. RESULTS: Our methods provide reliable stimulation in animals without the need for restraint or sedation. We see little aversive effects of support placement and stimulation. Computational models provide evidence that moving the coil support location can be utilized to target major stimulation sites in humans and mice. SUMMARY OF FINDINGS WITH THIS METHOD: Animal models are key to optimizing brain stimulation parameters, but research relies on restraint or sedation for consistency in coil placement. The method described here provides a unique means for reliable targeted stimulation in freely moving animals. Research utilizing this method has uncovered changes in biochemical and animal behavioral measurements as a function of brain stimulation. CONCLUSIONS: The majority of research on magnetic stimulation focuses on anterior regions. Given the substantial network connectivity throughout the brain, it is critical to develop a reliable method for stimulating different regions. The method described here can be utilized to better inform clinical trials about optimal treatment localization, stimulation intensity and number of treatment sessions, and provides a motivation for exploring posterior brain regions for both mice and humans.

Dimensions of Craving Interact with COMT Genotype to Predict Relapse in Individuals with Alcohol Use Disorder Six Months after Treatment.

(1) Background: Alcohol use disorder (AUD) is associated with poor medical, psychological, and psychosocial outcomes and approximately 60% of individuals with AUD relapse six months after treatment. Craving is a core aspect of AUD and associated with high risk of relapse. One promising avenue to improve outcomes may be in understanding the relationship between COMT genotype, craving, and treatment outcomes. (2) Methods: To this end, we assessed craving, recent drinking history, and impulsivity in 70 individuals with AUD undergoing a standard course of treatment at a regional Veteran Affairs (VA) medical center. Saliva samples were collected to determine COMT genotype. In this prospective observational study, participants were followed for six months to determine who went on to relapse after treatment. (3) Results: Results revealed a significant interaction between craving and catechol-O-methyltransferse (COMT) genotype in predicting relapse. Post hoc exploratory analyses indicated that Met/Met homozygotes reported the highest levels of craving, and craving was associated with recent drinking history. Among Val/Val homozygotes, who had higher rates of relapse, craving was associated with impulsivity. (4) Conclusions: These associations highlight that specific profiles of psychological and biological factors may be important in understanding which individuals are at highest risk of relapse following treatment. Future studies that build on these findings are warranted.

Perfluoroalkyl chemicals in neurological health and disease: Human concerns and animal models.

Perfluoroalkyl acids (PFAAs) are man-made organic pollutants that are found ubiquitously in the environment and may impact human health. Here, we review the published literature concerning PFAA impacts on neurobiological, neuroendocrine, and neurobehavioral outcomes. We find that there are many mechanisms through which PFAAs may enter the brain and interact with biochemical endpoints to impact neurological function. These results are supported by epidemiological evidence in humans and experimental evidence in animals that demonstrate numerous and varied PFAA impacts on the nervous system. However, the methods commonly used in animal models of PFAA exposure result in durations of exposure and serum PFAA concentrations in blood that may not appropriately mimic human absorption, distribution, metabolism, and excretion. If animal models lack validity, confidence in mechanistic inferences regarding PFAA exposure and brain function is reduced, limiting these studies' utility. Finally, we end by suggesting some potential impacts of PFAA exposure in human neurological health and disease states whose associations may not readily present themselves in the epidemiological literature.

Sleep-wake disorders in Alzheimer's disease: further genetic analyses in relation to objective sleep measures.

This paper presents updated analyses on the genetic associations of sleep disruption in individuals with Alzheimer's disease (AD). We published previously a study of the association between single nucleotide polymorphisms (SNPs) found in eight genes related to circadian rhythms and objective measures of sleep-wake disturbances in 124 individuals with AD. Here, we present new relevant analyses using polygenic risk scores (PRS) and variable number tandem repeats (VNTRs) enumerations. PRS were calculated using the genetic data from the original participants and relevant genome wide association studies (GWAS). VNTRs for the same circadian rhythm genes studied with SNPs were obtained from a separate cohort of participants using whole genome sequencing (WGS). Objectively (wrist actigraphy) determined wake after sleep onset (WASO) was used as a measure of sleep disruption. None of the PRS were associated with sleep disturbance. Computer analyses using VNTRseek software generated a total of 30 VNTRs for the circadian-related genes but none appear relevant to our objective sleep measure. In addition, of 71 neurotransmitter function-related genes, 29 genes had VNTRs that differed from the reference VNTR, but it was not clear if any of these might affect circadian function in AD patients. Although we have not found in either the current analyses or in our previous published analyses of SNPs any direct linkages between identified genetic factors and WASO, research in this area remains in its infancy.

Objective Classification of mTBI Using Machine Learning on a Combination of Frontopolar Electroencephalography Measurements and Self-reported Symptoms.

BACKGROUND: The reliable diagnosis of a mild traumatic brain injury (mTBI) is a pervasive problem in sports and in the military. The frequency and severity of each occurrence, while difficult to quantify, may impact long term cognitive function and quality of life. Despite the new revelations concerning brain disfunction from head injuries, individuals still feel pressure to remain on the field despite a debilitating injury. In this study, we evaluated the accuracy of a system that could be employed on the sidelines or in the locker room to provide an immediate objective mTBI assessment. METHODS: Participants consisted of 38 individuals with a recent mTBI and 47 controls with no history of mTBI within the last 5 years. Participants were administered a simple symptom questionnaire, behavioral tests, and resting state EEG was measured using three frontopolar electrodes. An advanced machine learning algorithm called boosting was utilized to classify subjects into either injured or controls using power spectral densities on 1-min of resting EEG and the symptom questionnaire. RESULTS: Results based on leave-one-out cross-validation revealed that the addition of EEG measurements boosted the accuracy to approximately 91 ± 2% compared to 82 ± 4% from the symptom questionnaire alone. CONCLUSION: This study demonstrated the potential benefit of including EEG measurements to diagnose suspected brain injury patients. This is a step toward accurate and objective classification measurements that can be implemented on the field as a future injury assessment tool.

Assessing disease-modifying effects of norepinephrine in Down syndrome and Alzheimer's disease.

Building upon the knowledge that a number of important brain circuits undergo significant degeneration in Alzheimer's disease, numerous recent studies suggest that the norepinephrine-ergic system in the brainstem undergoes significant alterations early in the course of both Alzheimer's disease and Down syndrome. Massive projections from locus coeruleus neurons to almost the entire brain, extensive innervation of brain capillaries, and widespread distribution of noradrenergic receptors enable the norepinephrine-ergic system to play a crucial role in neural processes, including cognitive function. These anatomical and functional characteristics support the role of the norepinephrine-ergic system as an important target for developing new therapies for cognitive dysfunction. Careful neuropathological examinations using postmortem samples from individuals with Alzheimer's disease have implicated the role of the norepinephrine-ergic system in the etiopathogenesis of Alzheimer's disease. Furthermore, numerous studies have supported the existence of a strong interaction between norepinephrine-ergic and neuroimmune systems. We explore the interaction between the two systems that could play a role in the disease-modifying effects of norepinephrine in Alzheimer's disease and Down syndrome.