Joerg R. Leheste

Distribution Analysis of Deacetylase SIRT1 in Rodent and Human Nervous Systems

Sirtuins function with other biogenic molecules to promote adaptation to caloric restriction in a broad spectrum of eukaryotic species. Sirtuin pathways also converge in the mammalian brain where they appear to protect neurons from nutrient stress. However, few anatomical studies on sirtuins (e.g., SIRT1) are available, particularly those detailing the spatial distribution and subcellular localization pattern of SIRT1 in the brain parenchyma. Here, we report the characterization of a panel of SIRT1‐specific antibodies within rodent (i.e., rat and mouse) and human central nervous systems. Immunocytochemical and Western blot analyses indicate that the subcellular localization of SIRT1 is predominantly nuclear throughout the rodent brain and spinal cord. A similar subcellular distribution pattern of SIRT1 was detected in human central nervous system material. SIRT1 is ubiquitously present in areas of the brain especially susceptible to age‐related neurodegenerative states (e.g., the prefrontal cortex, hippocampus and basal ganglia). Further, we show no apparent species‐specific differences in the subcellular localization pattern of rodent versus human SIRT1. Finally, we identify the chemical phenotype of SIRT1‐containing neurons in a number of brain sites that are strongly compromised by aging. These data provide additional and important anatomical findings for the role of SIRT1 in the mammalian brain and suggest that SIRT1 pathways are broadly distributed in neurons most susceptible to senescence injury. Activating endogenous sirtuin pathways may, therefore, offer a therapeutic approach to delay and/or treat human age‐related diseases. Anat Rec, 2010.

A behavioral and molecular analysis of ketamine in zebrafish

Ketamine exerts powerful anesthetic, psychotic, and antidepressant effects in both healthy volunteers and clinically depressed patients. Although ketamine targets particular glutamate receptors, there is a dearth of evidence for additional, alternative molecular substrates for the behavioral actions of this N‐methyl‐D‐aspartate (NMDA) receptor antagonist drug. Here, we provide behavioral and molecular evidence for the actions of ketamine using a new vertebrate model for psychiatric disorders: the zebrafish. Subanesthetic doses of ketamine produced a variety of abnormal behaviors in zebrafish that were qualitatively analogous to those previously measured in humans and rodents treated with drugs that produce transient psychosis. In addition, we revealed that the transcription factor Phox2b is a molecular substrate for the actions of ketamine, particularly during periods of hypoxic stress. Finally, we also show that SIRT1, a histone deacetylase widely recognized for its link to cell survival is also affected by hypoxia crises. These results establish a relevant assay system in which the effects of psychotomimetic drugs can rapidly be assessed, and provide a plausible and novel neuronal mechanism through which ketamine affects critical sensory circuits that monitor breathing behavior. Synapse, 2011.

Silent information regulator 1 mediates hippocampal plasticity through presenilin1

Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase directly implicated in protecting a wide range of organisms against internal and external metabolic insults. However, the identification of SIRT1-specific DNA targets that confer such protection have remained elusive. Using human cells, we show that SIRT1 binds to, and transcriptionally regulates, a gene locus encoding presenilin1 (PSEN1), a protein intrinsically involved in the function of the γ-secretase protein complex. We also demonstrate that rats fed with resveratrol exhibit a significant increase in sirt1 and psen1 expression. Finally, dietary consumption of resveratrol also leads to an enhanced proliferative state of neuronal stem cells in the rat hippocampus. Our findings reveal a strong link between resveratrol-dependent SIRT1 signaling and hippocampal plasticity in the mammalian brain.

A ChIP-cloning approach linking SIRT 1 to transcriptional modification of DNA targets

The mammalian protein deacetylase SIRT1 (sirtuin1) is widely recognized for its link to calorie restriction and longevity. SIRT1 not only modulates the function of protein targets such as p53 or NFkappaB, but it also affects gene transcription by causing hypoacetylation of associated nucleosomal histones. However, the identification of SIRT1-specific DNA targets that confer chromosomal stability and cell longevity have remained elusive. Here, we report the usefulness of a ChIP-cloning approach for the identification of an endogenous DNA target intimately linked with SIRT1 activity. Using the aforementioned technique, we identified a gene encoding the neuro-oncological ventral antigen2 (nova2) as a SIRT1 target. Nova2 regulates the alternative splicing of scn1a, which encodes the alpha-subunit of a neuronal sodium channel targeted by antiepileptic drugs. This finding demonstrates that ChIP-cloning is an innovative approach for the identification of SIRT1-specific DNA targets.

Distribution maps of d-dopachrome tautomerase in the mouse brain

D-Dopachrome tautomerase is an enzyme related by amino acid sequence and catalytic activity to macrophage migration inhibitory factor. Both of these small molecules are pro-inflammatory cytokines mediating broad innate immune responses. Although it is well established that the gene product of D-dopachrome tautomerase is widely expressed in liver and kidney cells, no study has mapped the distribution pattern of this tautomeric enzyme in the mammalian nervous system. Here, we address this void by characterizing the cellular localization of D-dopachrome tautomerase in the adult mouse brain. Two well-characterized polyclonal antibodies were used for Western blotting and immunohistochemical localization of the endogenous tautomeric enzyme. Our results show that D-dopachrome tautomerase is present throughout the brain parenchyma with a large fraction of heterogeneous interneurons harboring a stable and robust expression of the enzyme. These data point to a potential involvement of D-dopachrome tautomerase activity in the mature mouse brain, and suggest some functional and evolutionary relationship between innate immunity and tautomerization of D-dopachrome in mammalian species.D-Dopachrome tautomerase is an enzyme related by amino acid sequence and catalytic activity to macrophage migration inhibitory factor. Both of these small molecules are pro-inflammatory cytokines mediating broad innate immune responses. Although it is well established that the gene product of D-dopachrome tautomerase is widely expressed in liver and kidney cells, no study has mapped the distribution pattern of this tautomeric enzyme in the mammalian nervous system. Here, we address this void by characterizing the cellular localization of D-dopachrome tautomerase in the adult mouse brain. Two well-characterized polyclonal antibodies were used for Western blotting and immunohistochemical localization of the endogenous tautomeric enzyme. Our results show that D-dopachrome tautomerase is present throughout the brain parenchyma with a large fraction of heterogeneous interneurons harboring a stable and robust expression of the enzyme. These data point to a potential involvement of D-dopachrome tautomerase activity in the mature mouse brain, and suggest some functional and evolutionary relationship between innate immunity and tautomerization of D-dopachrome in mammalian species.

The Glymphatic-Lymphatic Continuum: Opportunities for Osteopathic Manipulative Medicine.

The brain has long been thought to lack a lymphatic drainage system. Recent studies, however, show the presence of a brain-wide paravascular system appropriately named the glymphatic system based on its similarity to the lymphatic system in function and its dependence on astroglial water flux. Besides the clearance of cerebrospinal fluid and interstitial fluid, the glymphatic system also facilitates the clearance of interstitial solutes such as amyloid-β and tau from the brain. As cerebrospinal fluid and interstitial fluid are cleared through the glymphatic system, eventually draining into the lymphatic vessels of the neck, this continuous fluid circuit offers a paradigm shift in osteopathic manipulative medicine. For instance, manipulation of the glymphatic-lymphatic continuum could be used to promote experimental initiatives for nonpharmacologic, noninvasive management of neurologic disorders. In the present review, the authors describe what is known about the glymphatic system and identify several osteopathic experimental strategies rooted in a mechanistic understanding of the glymphatic-lymphatic continuum.

Neurodevelopmental Malformations of the Cerebellar Vermis in Genetically Engineered Rats

The cerebellar vermis is particularly vulnerable to neurodevelopmental malformations in humans and rodents. Sprague–Dawley, and Long–Evans rats exhibit spontaneous cerebellar malformations consisting of heterotopic neurons and glia in the molecular layer of the vermis. Malformations are almost exclusively found along the primary fissure and are indicative of deficits of neuronal migration during cerebellar development. In the present report, we test the prediction that genetically engineered rats on Sprague–Dawley or Long–Evans backgrounds will also exhibit the same cerebellar malformations. Consistent with our hypothesis, we found that three different transgenic lines on two different backgrounds had cerebellar malformations. Heterotopia in transgenic rats had identical cytoarchitecture as that observed in wild-type rats including altered morphology of Bergmann glia. In light of the possibility that heterotopia could affect results from behavioral studies, these data suggest that histological analyses be performed in studies of cerebellar function or development when using genetically engineered rats on these backgrounds in order to have more careful interpretation of experimental findings.

Glutamate-Based Drugs for the Treatment of Clinical Depression

Clinical depression is a chronic, recurrent mood disorder that causes significant disability and disease burden throughout the world. Not surprisingly, there is an enormous demand for finding (a) appropriate medications and devices for treating the clinical symptoms and (b) the underlying molecular mechanisms of the disease. Currently, most therapeutic treatments of depression indirectly target the serotonin and norepinephrine systems of the brain, as these neurotransmitters have long been considered promising and mechanistically relevant to the etiology of mood disorders. However, selective serotonin reuptake inhibitors such as sertraline, fluoxetine and paroxetine do not always substantially improve clinical outcome, and when they do show efficacy, it takes weeks of treatment to achieve an appreciable clinical effect. These observations suggest that a serotonin and norepinephrine hypothesis of depression is incomplete at best, and that novel, rapid onset therapeutic options for depression must be considered. In this review, we highlight several potential new drugs for clinical depression based on recent discoveries about the neurotransmitter glutamate and its family of receptors. Moreover, we discuss the possibility that glutamate-based antidepressant drugs might affect covalent histone modifications including acetylation in areas of the brain (e.g., pre-frontal cortex, hippocampus) thought to be relevant for the pathogenesis of affective disorders. If so, histone hyperacetylation and thus chromatin remodeling might be important regulatory mechanisms underlying the effects of ketamine and other N-Methyl-D-Aspartate receptor antagonist drugs. Chromatin remodeling may represent a non-serotonin/norepinephrine therapeutic strategy for treatment of clinical depression, a strategy that may also be appropriate in the context of drug discovery and drug development.

Caenorhabditis elegans as an undergraduate educational tool for teaching RNAi

Discovery of RNA‐mediated interference (RNAi) is widely recognized as one of the most significant molecular biology breakthroughs in the past 10 years. There is a need for science educators to develop teaching tools and laboratory activities that demonstrate the power of this new technology and help students to better understand the RNAi process. C. elegans is an ideal model organism for the undergraduate laboratory because of the simplicity of worm maintenance, its well‐studied genetic background, and the fact that it can be employed as a model organism in laboratory environments where vertebrate research is restricted. Certain unique features of C. elegans make it a very suitable organism for RNAi studies. Specifically, nematode strains highly sensitive to RNAi are readily available from public sources, and RNAi induction by a feeding method is an uncomplicated procedure that lends itself readily as an educational tool. In this article, we provide a detailed depiction of the use of C. elegans as an RNAi educational tool, describing two separate RNAi‐based experiments. One is a qualitative experiment where students can examine the effects of knocking down the unc‐22 gene involved in the regulation of muscle contraction, which results in a “twitching” phenotype. The other experiment is a quantitative RNAi experiment, where students measure the effect of knocking down the lsy‐2 gene involved in neuronal development. Although these experiments are designed for a college‐level study, nematode research projects can also be accomplished in secondary school facilities.