targeted-next-generation-sequencing-bioinformatics-pipeline-to

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

McMaster University

In spite of the significant resources devoted to medical education at all levels, the study of educational process has received relatively little attention. Most studies of learning have been observational, with little reference to recent advances in the neurobiology of learning and memory. In this article, the author briefly reviews developments in our understanding of the neurobiology of learning and memory and suggests ways they could contribute to the future evolution of medical education.

Bench to bedside in medical education.

Long-case patient-based examinations previously formed the basis of summative competency testing in physician certification examinations. These exams were found to be unreliable and have fallen from favor. During the authors' deliberation of the long case in the neurology certification examinations of the Royal College of Physicians and Surgeons of Canada, they considered the examination context and concluded that the appropriate psychometric analysis of the exams is highly contingent on the context. The examination context underlying certification examinations has evolved considerably; within a different context, a more cohesive test system based on a quality assurance framework could better manage substantive psychometric issues around case specificity, comprehensiveness, reliability, and compensability. These arguments are in small part psychometric, but are mostly philosophical and have relevance to the profession and the public.

Contextual considerations in summative competency examinations: relevance to the long case.

Remediation of incompetent physicians has proven difficult and sometimes impossible. The authors wished to determine whether such physicians had neuropsychological impairment sufficient to explain their incompetence and their failure to improve after remedial continuing medical education (CME).

Competence and cognitive difficulty in physicians: a follow-up study.

The primary cilium is a solitary organelle important in cellular signaling, that projects from the cell surface of most growth-arrested or post-mitotic cells including neurons in the central nervous system. We hypothesized that primary cilial dysfunction might play a role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), and as a first step, report on the prevalence of primary cilial markers on cultured motor neurons from the lumbar spinal cord of embryonic wildtype (WT) and transgenic G93A SOD1 mice, and on motor neurons in situ in the lumbar spinal cord.

Adenylyl cyclase type 3, a marker of primary cilia, is reduced in primary cell culture and in lumbar spinal cord in situ in G93A SOD1 mice.

We have previously demonstrated that primary cilia on spinal motor neurons are reduced in G93A SOD1 (mSOD) mice, a mouse model of amyotrophic lateral sclerosis (ALS). Sonic hedgehog (Shh) signaling involves the primary cilium and Shh has been shown to be cytoprotective in models of other neurodegenerative diseases. Thus, the Shh signaling pathway may bear further study in ALS. Accordingly, we established that interference with the Shh pathway (with the Shh antagonist cyclopamine or with miRNA 3245p) sensitized HT22 cells, while augmentation of the Shh pathway (with Shh or the Shh agonist purmorphamine) protected cells against hydrogen peroxide (H₂O₂) challenge. We ectopically expressed mSOD, human wild-type SOD1 (wtSOD), or an empty vector in HT22 cells. Compared to empty vector, wtSOD decreased cell death and mSOD increased cell death in response to H₂O₂ challenge. Treatment with cyclopamine or miRNA 3245p sensitized all three transfections to H₂O₂ challenge. Treatment with recombinant human Shh or purmorphamine decreased cell death after H₂O₂ challenge, an effect more pronounced in mSOD cells. Compared with empty vector, overexpression of wtSOD increased Shh and Gli transcript levels and increased activity in a Gli-responsive reporter assay. Overexpression of mSOD did not change Shh transcript levels, but decreased Gli transcript levels, especially Gli3, and reduced activity in a Gli reporter assay. These results suggest that overexpression of mSOD but not wtSOD reduces signaling in the Shh pathway and renders mSOD cells more susceptible to H₂O₂ challenge, and that treatment with Shh or Shh agonists is cytoprotective to mSOD cells. Shh or Shh agonists merit further consideration as potential therapy in ALS.

Sonic hedgehog is cytoprotective against oxidative challenge in a cellular model of amyotrophic lateral sclerosis.

Methylene blue (MB) is a drug with a long history and good safety profile, and with recently-described features desirable in a treatment for ALS.

Lack of effect of methylene blue in the SOD1 G93A mouse model of amyotrophic lateral sclerosis.

The developmental morphogen sonic hedgehog (Shh) may continue to play a trophic role in the support of terminally-differentiated motor neurons, of potential relevance to motor neuron disease. In addition, it may support the proliferation and differentiation of endogenous stem cells along motor neuronal lineages. As such, we have examined the trophic and proliferative effects of Shh supplementation or Shh antagonism in embryonic spinal cord cell cultures derived from wildtype or G93A SOD1 mice, a mouse model of amyotrophic lateral sclerosis.

Trophic and proliferative effects of Shh on motor neurons in embryonic spinal cord culture from wildtype and G93A SOD1 mice.

Several studies have illustrated that the polymerase gamma mutator (PolG) mice have reduced mitochondrial content secondary to systemic mitochondrial dysfunction, and subsequently a lower capacity to perform aerobic respiration and endurance exercise. We sought to delineate the extent of glycolysis as a means of energy production in the PolG mice in the absence of optimal mitochondrial function. PolG mice display an enhanced reliance on glycolysis as compared to their wild-type counterparts. This is evident by the resting hypoglycemia, higher PFK content, and elevated plasma lactate levels in the PolG mice. In vitro experiments provide further proof that PolG derived dermal fibroblasts have a higher rate of, and capacity for, glycolysis. PolG mice also have enhanced capacity to perform hepatic gluconeogenesis that is likely enhancing the Cori cycle capacity.

Polymerase gamma mutator mice rely on increased glycolytic flux for energy production.

NeuN is a nuclear protein expressed exclusively in mature neurons and has served for many years as a reliable neuronal marker in immunohistochemical labeling studies. In 2009, NeuN was identified as Fox3, one of three closely related RNA binding proteins important in pre-mRNA splicing. During the course of a previous study using G93A SOD1 mice, a model of amyotrophic lateral sclerosis (ALS), we observed that NeuN was significantly redistributed to the cytosol. Since altered splicing may be important in the pathogenesis of ALS, we compared the localization (predominantly nuclear or cytosolic) of all three Fox proteins in the lumbar spinal cord of wild-type and G93A SOD1 mice before and after the development of clinical signs of disease. The Fox proteins regulate their own splicing, and we also examined the major Fox protein isoforms in nuclear and cytosolic fractions of lumbar spinal cord by Western blotting. We report here that Fox3 and Fox2 undergo a major cytosolic relocalization in this ALS model that increases with age and that is associated with progressive alterations in the splicing profiles of all three Fox proteins.

Cytosolic localization of Fox proteins in motor neurons of G93A SOD1 mice.

The developmental morphogen Sonic hedgehog (Shh) may continue to play a sustaining role in adult motor neurons, of potential relevance to motor neuron diseases including amyotrophic lateral sclerosis. The Shh signaling pathway is incompletely understood and interactions with other signaling pathways are possible. We focus here on Notch, and first show that there is an almost linear reduction in light output from a Gli reporter in Shh Light II cells in the presence of increasing concentrations of the Notch inhibitor DAPT (r=0.982). Second, in the spinal cord of mutant superoxide dismutase mice, but not control mice, a key marker of Notch signaling changes with age. Before the onset of clinical signs, the Notch intracellular domain is expressed predominantly in motor neurons, but by 125 days of age, Notch intracellular domain expression is markedly reduced in motor neurons and increased in neighboring astroglia. Third, there is a parallel reduction in Gli protein expression in mutant superoxide dismutase mouse spinal motor neurons, consistent with the observed reduction in Notch signaling and also a redistribution of Gli away from the nucleus. Thus, there is a reduction in motor neuronal Notch signaling and associated changes in Shh signaling, occurring coincidentally with disease expression, that may contribute toward the dysfunction and death of motor neurons in amyotrophic lateral sclerosis.

Crosstalk between Notch and Sonic hedgehog signaling in a mouse model of amyotrophic lateral sclerosis.

Sonic hedgehog (Shh) is a morphogen essential to the developing nervous system that continues to play an important role in adult life by contributing to cell proliferation and differentiation, maintaining blood-brain barrier integrity, and being cytoprotective against oxidative and excitotoxic stress, all features of importance in amyotrophic lateral sclerosis (ALS). ALS is a fatal disease characterized by selective loss of motor neurons due to poorly understood mechanisms. Evidence indicates that Shh might play an important role in ALS, and that Shh signaling might be also adversely affected in ALS. Since little is known about the functional status of Shh pathway in patients with ALS, we therefore sought to determine whether Shh protein levels or biological activity in cerebrospinal fluid (CSF) was less in ALS patients than controls, and whether these measures could be correlated with ALS disease severity and disease progression, and with other CSF analytes of biological interest in ALS. Comparing Shh levels in the CSF of normal controls (n = 13), neurological controls (n = 12), and ALS patients (n = 9) measured by ELISA, we found that CSF Shh levels were not different between controls and ALS patients. However, when assessing Shh biological activity in CSF using in vitro cell-based assays, which measure Shh activity as inducible Gli-driven luminescence, we found that in the presence of exogenous recombinant Shh or the Shh agonist, purmorphamine, the inducible activity of CSF was significantly augmented in the control groups as expected, but not in the ALS group, suggesting the presence of an inhibitor of Shh signaling in ALS CSF samples. Since purmorphamine acts on Smoothened, downstream of Shh and its receptor Patched, the inhibitory action is downstream of Smoothened. Our results also demonstrated that while the inhibitory effect of ALS CSF on Shh signaling did not correlate significantly with ALS disease characteristics, the levels of IL-1β and TNF-α did. In addition to being significantly elevated in ALS CSF, these cytokines negatively correlated with the disease duration, whereas GDF11 was a favorable predictor of ALS clinical score. We also found that TNF-α significantly inhibited Shh biological activity in vitro, potentially suggesting a novel role of TNF-α in ALS pathogenesis. Collectively, this is the first report demonstrating that Shh signaling in CSF of ALS patients is compromised.

Article	Year
Bench to bedside in medical education. Academic medicine : journal of the Association of American Medical Colleges\| PubMed ID: 10386093	1999
Contextual considerations in summative competency examinations: relevance to the long case. Academic medicine : journal of the Association of American Medical Colleges\| PubMed ID: 16306287	2005
Competence and cognitive difficulty in physicians: a follow-up study. Academic medicine : journal of the Association of American Medical Colleges\| PubMed ID: 16985357	2006
Adenylyl cyclase type 3, a marker of primary cilia, is reduced in primary cell culture and in lumbar spinal cord in situ in G93A SOD1 mice. BMC neuroscience\| PubMed ID: 21767396	2011
Sonic hedgehog is cytoprotective against oxidative challenge in a cellular model of amyotrophic lateral sclerosis. Journal of molecular neuroscience : MN\| PubMed ID: 21979788	2012
Lack of effect of methylene blue in the SOD1 G93A mouse model of amyotrophic lateral sclerosis. PloS one\| PubMed ID: 21998625	2011
Trophic and proliferative effects of Shh on motor neurons in embryonic spinal cord culture from wildtype and G93A SOD1 mice. BMC neuroscience\| PubMed ID: 24119209	2013
Polymerase gamma mutator mice rely on increased glycolytic flux for energy production. Mitochondrion\| PubMed ID: 25575636	2015
Cytosolic localization of Fox proteins in motor neurons of G93A SOD1 mice. Histochemistry and cell biology\| PubMed ID: 26724814	2016
Crosstalk between Notch and Sonic hedgehog signaling in a mouse model of amyotrophic lateral sclerosis. Neuroreport\| PubMed ID: 27984541	2017
Cerebrospinal fluid from patients with amyotrophic lateral sclerosis inhibits sonic hedgehog function. PloS one\| PubMed ID: 28170441	2017

John Turnbull

Biography

JoVE Journal Publications

Other Publications