Kevin K. Kumar

Age-related sperm DNA methylation changes are transmitted to offspring and associated with abnormal behavior and dysregulated gene expression.

Advanced paternal age (APA) has been shown to be a significant risk factor in the offspring for neurodevelopmental psychiatric disorders, such as schizophrenia and autism spectrum disorders. During aging, de novo mutations accumulate in the male germline and are frequently transmitted to the offspring with deleterious effects. In addition, DNA methylation during spermatogenesis is an active process, which is susceptible to errors that can be propagated to subsequent generations. Here we test the hypothesis that the integrity of germline DNA methylation is compromised during the aging process. A genome-wide DNA methylation screen comparing sperm from young and old mice revealed a significant loss of methylation in the older mice in regions associated with transcriptional regulation. The offspring of older fathers had reduced exploratory and startle behaviors and exhibited similar brain DNA methylation abnormalities as observed in the paternal sperm. Offspring from old fathers also had transcriptional dysregulation of developmental genes implicated in autism and schizophrenia. Our findings demonstrate that DNA methylation abnormalities arising in the sperm of old fathers are a plausible mechanism to explain some of the risks that APA poses to resulting offspring.

Genetic risk for Parkinson's disease correlates with alterations in neuronal manganese sensitivity between two human subjects.

Manganese (Mn) is an environmental risk factor for Parkinsons disease (PD). Recessive inheritance of PARK2 mutations is strongly associated with early onset PD (EOPD). It is widely assumed that the influence of PD environmental risk factors may be enhanced by the presence of PD genetic risk factors in the genetic background of individuals. However, such interactions may be difficult to predict owing to the complexities of genetic and environmental interactions. Here we examine the potential of human induced pluripotent stem (iPS) cell-derived early neural progenitor cells (NPCs) to model differences in Mn neurotoxicity between a control subject (CA) with no known PD genetic risk factors and a subject (SM) with biallelic loss-of-function mutations in PARK2 and family history of PD but no evidence of PD by neurological exam. Human iPS cells were generated from primary dermal fibroblasts of both subjects. We assessed several outcome measures associated with Mn toxicity and PD. No difference in sensitivity to Mn cytotoxicity or mitochondrial fragmentation was observed between SM and CA NPCs. However, we found that Mn exposure was associated with significantly higher reactive oxygen species (ROS) generation in SM compared to CA NPCs despite significantly less intracellular Mn accumulation. Thus, this report offers the first example of human subject-specific differences in PD-relevant environmental health related phenotypes that are consistent with pathogenic interactions between known genetic and environmental risk factors for PD.

The potential of induced pluripotent stem cells as a translational model for neurotoxicological risk

An important goal of neurotoxicological research is to provide relevant and accurate risk assessment of environmental and pharmacological agents for populations and individuals. Owing to the challenges of human subject research and the real possibility of species specific toxicological responses, neuronal lineages derived from human embryonic stem cells (hESCs) and human neuronal precursors have been offered as a potential solution for validation of neurotoxicological data from model organism systems in humans. More recently, with the advent of induced pluripotent stem cell (iPSC) technology, there is now the possibility of personalized toxicological risk assessment, the ability to predict individual susceptibility to specific environmental agents, by this approach. This critical advance is widely expected to facilitate analysis of cellular physiological pathways in the context of human neurons and the underlying genetic factors that lead to disease. Thus this technology opens the opportunity, for the first time, to characterize the physiological, toxicological, pharmacological and molecular properties of living human neurons with identical genetic determinants as human patients. Furthermore, armed with a complete clinical history of the patients, human iPSC (hiPSC) studies can theoretically compare patients and at risk groups with distinct sensitivities to particular environmental agents, divergent clinical outcomes, differing co-morbidities, and so forth. Thus iPSCs and neuronal lineages derived from them may reflect the unique genetic blueprint of the individuals from which they are generated. Indeed, iPSC technology has the potential to revolutionize scientific approaches to human health. However, before this overarching goal can be reached a number of technical and theoretical challenges must be overcome. This review seeks to provide a realistic assessment of hiPSC technology and its application to risk assessment and mechanistic studies in the area of neurotoxicology. We seek to identify, prioritize, and detail the primary hurdles that need to be overcome if personalized toxicological risk assessment using patient-derived iPSCs is to succeed.

A novel manganese-dependent ATM-p53 signaling pathway is selectively impaired in patient-based neuroprogenitor and murine striatal models of Huntington's disease

The essential micronutrient manganese is enriched in brain, especially in the basal ganglia. We sought to identify neuronal signaling pathways responsive to neurologically relevant manganese levels, as previous data suggested that alterations in striatal manganese handling occur in Huntingtons disease (HD) models. We found that p53 phosphorylation at serine 15 is the most responsive cell signaling event to manganese exposure (of 18 tested) in human neuroprogenitors and a mouse striatal cell line. Manganese-dependent activation of p53 was severely diminished in HD cells. Inhibitors of ataxia telangiectasia mutated (ATM) kinase decreased manganese-dependent phosphorylation of p53. Likewise, analysis of ATM autophosphorylation and additional ATM kinase targets, H2AX and CHK2, support a role for ATM in the activation of p53 by manganese and that a defect in this process occurs in HD. Furthermore, the deficit in Mn-dependent activation of ATM kinase in HD neuroprogenitors was highly selective, as DNA damage and oxidative injury, canonical activators of ATM, did not show similar deficits. We assessed cellular manganese handling to test for correlations with the ATM-p53 pathway, and we observed reduced Mn accumulation in HD human neuroprogenitors and HD mouse striatal cells at manganese exposures associated with altered p53 activation. To determine if this phenotype contributes to the deficit in manganese-dependent ATM activation, we used pharmacological manipulation to equalize manganese levels between HD and control mouse striatal cells and rescued the ATM-p53 signaling deficit. Collectively, our data demonstrate selective alterations in manganese biology in cellular models of HD manifest in ATM-p53 signaling.

PARK2 patient neuroprogenitors show increased mitochondrial sensitivity to copper

Poorly-defined interactions between environmental and genetic risk factors underlie Parkinsons disease (PD) etiology. Here we tested the hypothesis that human stem cell derived forebrain neuroprogenitors from patients with known familial risk for early onset PD will exhibit enhanced sensitivity to PD environmental risk factors compared to healthy control subjects without a family history of PD. Two male siblings (SM and PM) with biallelic loss-of-function mutations in PARK2 were identified. Human induced pluripotent stem cells (hiPSCs) from SM, PM, and four control subjects with no known family histories of PD or related neurodegenerative diseases were utilized. We tested the hypothesis that hiPSC-derived neuroprogenitors from patients with PARK2 mutations would show heightened cell death, mitochondrial dysfunction, and reactive oxygen species generation compared to control cells as a result of exposure to heavy metals (PD environmental risk factors). We report that PARK2 mutant neuroprogenitors showed increased cytotoxicity with copper (Cu) and cadmium (Cd) exposure but not manganese (Mn) or methyl mercury (MeHg) relative to control neuroprogenitors. PARK2 mutant neuroprogenitors also showed a substantial increase in mitochondrial fragmentation, initial ROS generation, and loss of mitochondrial membrane potential following Cu exposure. Our data substantiate Cu exposure as an environmental risk factor for PD. Furthermore, we report a shift in the lowest observable effect level (LOEL) for greater sensitivity to Cu-dependent mitochondrial dysfunction in patients SM and PM relative to controls, correlating with their increased genetic risk for PD.

Bone loss in anorexia nervosa: leptin, serotonin, and the sympathetic nervous system

Anorexia nervosa (AN), a disorder characterized by the refusal to sustain a healthy weight, has the highest mortality of any psychiatric disorder. This review presents a model of AN that ties together advances in our understanding of how leptin, serotonin, and hypogonadism are brought about in AN and how they influence bone mass. Serotonin (5‐hydroxytryptamine) is a key regulator of satiety and mood. The primary disturbance in AN results from alterations in serotonin signaling. AN patients suffer from serotonergic hyperactivity of Htr1a‐dependent pathways that causes dysphoric mood and promotes restrictive behavior. By limiting carbohydrate ingestion, anorexics decrease their serotonin levels. Reduced serotonergic signaling in turn suppresses appetite through Htr1a/2b, decreases dysphoric mood through Htr1a/2a, and activates the sympathetic nervous system (SNS) through Htr2c receptors in the ventromedial hypothalamus. Activation of the SNS decreases bone mass through β2‐adrenergic signaling in osteoblasts. Additional topics reviewed here include osteoblastic feedback of metabolism in anorexia, mechanisms whereby dietary changes exacerbate bone loss, the role of caloric restriction and Sirt1 in bone metabolism, hypothalamic hypogonadisms effects on bone mass, and potential treatments.

Untargeted metabolic profiling identifies interactions between Huntington's disease and neuronal manganese status

Manganese (Mn) is an essential micronutrient for development and function of the nervous system. Deficiencies in Mn transport have been implicated in the pathogenesis of Huntingtons disease (HD), an autosomal dominant neurodegenerative disorder characterized by loss of medium spiny neurons of the striatum. Brain Mn levels are highest in striatum and other basal ganglia structures, the most sensitive brain regions to Mn neurotoxicity. Mouse models of HD exhibit decreased striatal Mn accumulation and HD striatal neuron models are resistant to Mn cytotoxicity. We hypothesized that the observed modulation of Mn cellular transport is associated with compensatory metabolic responses to HD pathology. Here we use an untargeted metabolomics approach by performing ultraperformance liquid chromatography-ion mobility-mass spectrometry (UPLC-IM-MS) on control and HD immortalized mouse striatal neurons to identify metabolic disruptions under three Mn exposure conditions, low (vehicle), moderate (non-cytotoxic) and high (cytotoxic). Our analysis revealed lower metabolite levels of pantothenic acid, and glutathione (GSH) in HD striatal cells relative to control cells. HD striatal cells also exhibited lower abundance and impaired induction of isobutyryl carnitine in response to increasing Mn exposure. In addition, we observed induction of metabolites in the pentose shunt pathway in HD striatal cells after high Mn exposure. These findings provide metabolic evidence of an interaction between the HD genotype and biologically relevant levels of Mn in a striatal cell model with known HD by Mn exposure interactions. The metabolic phenotypes detected support existing hypotheses that changes in energetic processes underlie the pathobiology of both HD and Mn neurotoxicity.

Genomic Instability Associated with p53 Knockdown in the Generation of Huntington's Disease Human Induced Pluripotent Stem Cells.

Alterations in DNA damage response and repair have been observed in Huntington’s disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown.

Optimization of Fluorescence Assay of Cellular Manganese Status for High Throughput Screening

The advent of high throughput screening (HTS) technology permits identification of compounds that influence various cellular phenotypes. However, screening for small molecule chemical modifiers of neurotoxicants has been limited by the scalability of existing phenotyping assays. Furthermore, the adaptation of existing cellular assays to HTS format requires substantial modification of experimental parameters and analysis methodology to meet the necessary statistical requirements. Here we describe the successful optimization of the Cellular Fura‐2 Manganese Extraction Assay (CFMEA) for HTS. By optimizing cellular density, manganese (Mn) exposure conditions, and extraction parameters, the sensitivity and dynamic range of the fura‐2 Mn response was enhanced to permit detection of positive and negative modulators of cellular manganese status. Finally, we quantify and report strategies to control sources of intra‐ and interplate variability by batch level and plate‐geometric level analysis. Our goal is to enable HTS with the CFMEA to identify novel modulators of Mn transport.

Hair sparing does not compromise real-time magnetic resonance imaging guided stereotactic laser fiber placement for temporal lobe epilepsy

Pre-operative scalp shaving is conventionally thought to simplify postoperative cranial wound care, lower the rate of wound infections, and ease optimal incision localization. Over the past few decades, some neurosurgeons have refrained from scalp shaving in order to improve patient satisfaction with brain surgery. However, this hair-sparing approach has not yet been explored in the growing field of magnetic resonance-guided laser interstitial thermal therapy (MRgLITT). This study investigated the initial impact of a no-shave technique on post-operative wound infection rate as well as on entry and target accuracy in MRgLITT for mesial temporal epilepsy. Eighteen patients selected by the Stanford Comprehensive Epilepsy Program between November 2015 and August 2017 were included in the study. All patients underwent functional selective amygdalohippocampotomies using MRgLITT entirely within a diagnostic MRI suite. No hair was removed and no additional precautions were taken for hair or scalp care. Otherwise, routine protocols for surgical preparations and wound closure were followed. The study was performed under approval from Stanford Universitys Internal Review Board (IRB-37830). No post-operative wound infections or erosions occurred for any patient. The mean entry point error was 2.87 ± 1.3 mm and the mean target error was 1.0 ± 0.9 mm. There have been no other complications associated with this hair-sparing approach. The studys results suggest that hair sparing in MRgLITT surgery for temporal epilepsy does not increase the risk of wound complications or compromise accuracy. This preferred cosmetic approach may thus appeal to epilepsy patients considering such interventions.