45 research outputs found
Dysregulated ERK/MAPK Signaling in RASopathy Animal Model Systems Leads to a Decrease in mTOR Expression and Activation of Translational Machinery
No full textabstract: The RAS/MAPK (RAS/Mitogen Activated Protein Kinase) pathway is a highly conserved, canonical signaling cascade that is highly involved in cellular growth and proliferation as well as cell migration. As such, it plays an important role in development, specifically in development of the nervous system. Activation of ERK is indispensable for the differentiation of Embryonic Stem Cells (ESC) into neuronal precursors (Li z et al, 2006). ERK signaling has also shown to mediate Schwann cell myelination of the peripheral nervous system (PNS) as well as oligodendrocyte proliferation (Newbern et al, 2011). The class of developmental disorders that result in the dysregulation of RAS signaling are known as RASopathies. The molecular and cell-specific consequences of these various pathway mutations remain to be elucidated. While there is evidence for altered DNA transcription in RASopathies, there is little work examining the effects of the RASopathy-linked mutations on protein translation and post-translational modifications in vivo. RASopathies have phenotypic and molecular similarities to other disorders such as Fragile X Syndrome (FXS) and Tuberous Sclerosis (TSC) that show evidence of aberrant protein synthesis and affect related pathways. There are also well-defined downstream RAS pathway elements involved in translation. Additionally, aberrant corticospinal axon outgrowth has been observed in disease models of RASopathies (Xing et al, 2016). For these reasons, this present study examines a subset of proteins involved in translation and translational regulation in the context of RASopathy disease states. Results indicate that in both of the tested RASopathy model systems, there is altered mTOR expression. Additionally the loss of function model showed a decrease in rps6 activation. This data supports a role for the selective dysregulation of translational control elements in RASopathy models. This data also indicates that the primary candidate mechanism for control of altered translation in these modes is through the altered expression of mTOR
Cell
No full textDeletion of UBE3A causes the neurodevelopmental disorder Angelman syndrome (AS), while duplication or triplication of UBE3A is linked to autism. These genetic findings suggest that the ubiquitin ligase activity of UBE3A must be tightly maintained to promote normal brain development. Here, we found that protein kinase A (PKA) phosphorylates UBE3A in a region outside of the catalytic domain at residue T485 and inhibits UBE3A activity toward itself and other substrates. A de novo autism-linked missense mutation disrupts this phosphorylation site, causing enhanced UBE3A activity in vitro, enhanced substrate turnover in patient-derived cells, and excessive dendritic spine development in the brain. Our study identifies PKA as an upstream regulator of UBE3A activity and shows that an autism-linked mutation disrupts this phosphorylation control. Moreover, our findings implicate excessive UBE3A activity and the resulting synaptic dysfunction to autism pathogenesis.5P41EB002025/EB/NIBIB NIH HHS/United StatesDP1 ES024088/ES/NIEHS NIH HHS/United StatesDP1ES024088/DP/NCCDPHP CDC HHS/United StatesP01 GM103723/GM/NIGMS NIH HHS/United StatesP01-GM103723/GM/NIGMS NIH HHS/United StatesP30 HD003110/HD/NICHD NIH HHS/United StatesP30 NS045892/NS/NINDS NIH HHS/United StatesP30NS045892/NS/NINDS NIH HHS/United StatesP41 EB002025/EB/NIBIB NIH HHS/United StatesR01 MH093372/MH/NIMH NIH HHS/United StatesR01 NS031768/NS/NINDS NIH HHS/United StatesR01 NS085093/NS/NINDS NIH HHS/United StatesR01MH093372/MH/NIMH NIH HHS/United StatesR01NS085093/NS/NINDS NIH HHS/United StatesT32HD040127/HD/NICHD NIH HHS/United StatesU54 HD079124/HD/NICHD NIH HHS/United StatesU54HD079124/HD/NICHD NIH HHS/United States2016-08-13T00:00:00Z26255772PMC453784
Signaling Endosomes Trigger Synapse Assembly
No full textIn this issue of Neuron, Sharma et al. report that target-derived NGF exerts long-distance control of postsynaptic assembly. A major advance is the discovery that TrkA signaling endosomes are retrogradely transported to neuronal dendrites and directly trigger the clustering of postsynaptic density proteins
Mechanisms of Recovery from Chronic Stress
No full textabstract: Chronic stress results in functional and structural changes to the hippocampus. Decades of research has led to insights into the mechanisms underlying the chronic stress-induced deficits in hippocampal-mediated cognition and reduction of dendritic complexity of hippocampal neurons. Recently, a considerable focus of chronic stress research has investigated the mechanisms behind the improvements in hippocampal mediated cognition when chronic stress ends and a post-stress rest period is given. Consequently, the goal of this dissertation is to uncover the mechanisms that allow for spatial ability to improve in the aftermath of chronic stress. In chapter 2, the protein brain derived neurotrophic factor (BDNF) was investigated as a mechanism that allows for spatial ability to show improvements following the end of chronic stress. It was found that decreasing the expression of BDNF in the hippocampus prevented spatial memory improvements following a post-stress rest period. Chapter 3 was performed to determine whether hippocampal CA3 apical dendritic complexity requires BDNF to show improvements following a post-stress rest period, and whether a receptor for BDNF, TrkB, mediates the improvements of spatial ability and dendritic complexity in a temporal manner, i.e. during the rest period only. These experiments showed that decreased hippocampal BDNF expression prevented improvements in dendritic complexity, and administration of a TrkB antagonist during the rest period also prevented the improvements in spatial ability and dendritic complexity. In chapter 4, the role of the GABAergic system on spatial ability following chronic stress and a post-stress rest period was investigated. Following chronic stress, it was found that male rats showed impairments on the acquisition phase of the RAWM and this correlated with limbic glutamic acid decarboxylase, a marker for GABA. In chapter 5, a transgenic mouse that expresses a permanent marker on all GABAergic interneurons was used to assess the effects of chronic stress and a post-stress rest period on hippocampal GABAergic neurons. While no changes were found on the total number of GABAergic interneurons, specific subtypes of GABAergic interneurons were affected by stressor manipulations. Collectively, these studies reveal some mechanisms behind the plasticity seen in the hippocampus in response to a post-stress rest period.Dissertation/ThesisDoctoral Dissertation Psychology 201
The Therapeutic Potential of Serotonin 1B Receptor Agonists for Treating Psychostimulant Use Disorders
No full textabstract: Serotonin 1B receptor (5-HT1BR) agonists enhance cocaine intake in rats during daily self-administration (SA) sessions, yet decrease cocaine intake after prolonged abstinence. The goal of my dissertation was to examine if 5-HT1BRs are suitable targets for treatment development to attenuate psychostimulant intake. I first investigated if 5-HT1BR agonist effects that had been observed with cocaine generalize across psychostimulants, i.e., methamphetamine. Rats trained to self-administer methamphetamine received either CP 94,253 or the clinically-available but less selective 5-HT1D/1BR agonist, zolmitriptan, prior to tests for effects on SA both before and after a 21-day abstinence period. I found that CP 94,253 and zolmitriptan decreased the reinforcing and incentive motivational effects of methamphetamine, regardless of abstinence, unlike the pre-abstinence increase in cocaine SA observed previously with 5-HT1BR agonists. The attenuating effects of CP 94,253 on methamphetamine were antagonized in a 5-HT1BR-mediated manner. Subsequently, I investigated the efficacy and mechanism involved in effects of zolmitriptan on cocaine SA in male and female rats. Rats trained to self-administer cocaine received zolmitriptan prior to tests for effects on SA before a 21-day abstinence period. I found that zolmitriptan decreased cocaine intake in both sexes regardless of abstinence and without altering sucrose intake. I further demonstrated that the zolmitriptan effects on cocaine SA were mediated by both 5-HT1BRs and 5-HT1DRs. Finally, I examined if the abstinence-induced decrease in cocaine intake observed with the selective 5-HT1BR agonist, CP 94,253, persists during relapse after abstinence or reverts to enhancing cocaine intake, similar to effects observed without an abstinence period. Rats trained to self-administer cocaine resumed daily cocaine SA sessions after the forced abstinence period to investigate the effects of CP 94,253 on cocaine relapse. I found that CP 94,253 attenuated cocaine intake in relapse tests, suggesting that the abstinence-dependent attenuation of CP 94,253 on cocaine SA remains even after resumption of daily cocaine intake. The findings suggest that 5-HT1BR agonists like CP 94,253 and zolmitriptan have clinical potential as treatments for psychostimulant use disorders.Dissertation/ThesisDoctoral Dissertation Neuroscience 202
Transcriptomic and Cellular Studies of Tail Regeneration in Saurian Reptiles
No full textabstract: Traumatic injury to the central nervous or musculoskeletal system in traditional amniote models, such as mouse and chicken, is permanent with long-term physiological and functional effects. However, among amniotes, the ability to regrow complex, multi-tissue structures is unique to non-avian reptiles. Structural regeneration is extensively studied in lizards, with most species able to regrow a functional tail. The lizard regenerated tail includes the spinal cord, cartilage, de novo muscle, vasculature, and skin, and unlike mammals, these tissues can be replaced in lizards as adults. These studies focus on the events that occur before and after the tail regrowth phase, identifying conserved mechanisms that enable functional tail regeneration in the green anole lizard, Anolis carolinensis. An examination of coordinated interactions between peripheral nerves, Schwann cells, and skeletal muscle reveal that reformation of the lizard neuromuscular system is dependent upon developmental programs as well as those unique to the adult during late stages of regeneration. On the other hand, transcriptomic analysis of the early injury response identified many immunoregulatory genes that may be essential for inhibiting fibrosis and initiating regenerative programs. Lastly, an anatomical and histological study of regrown alligator tails reveal that regenerative capacity varies between different reptile groups, providing comparative opportunities within amniotes and across vertebrates. In order to identify mechanisms that limit regeneration, these cross-species analyses will be critical. Taken together, these studies serve as a foundation for future experimental work that will reveal the interplay between reparative and regenerative mechanisms in adult amniotes with translational implications for medical therapies.Dissertation/ThesisDifferentially Expressed Genes in the Early Regenerating Lizard TailGene Ontology of Differentially Expressed Genes in the Early Regenerating Lizard TailKEGG and Reactome Pathway Analysis of Differentially Expressed Genes in the Early Regenerating Lizard Tail3D Reconstruction of an Alligator Regenerated EndoskeletonLateral 2D Serial Sections of a Regenerated Alligator TailDoctoral Dissertation Biology 202
Disrupted Synaptic Transmission and Abnormal Short-term Synaptic Plasticity in an Angelman Syndrome Mouse Model
No full textabstract: Angelman syndrome (AS) is a neurodevelopmental disorder characterized by developmental delays, intellectual disabilities, impaired language and speech, and movement defects. Most AS cases are caused by dysfunction of a maternally-expressed E3 ubiquitin ligase (UBE3A, also known as E6 associated protein, E6-AP) in neurons. Currently, the mechanism on how loss-of-function of the enzyme influences the nervous system development remains unknown. We hypothesize that impaired metabolism of proteins, most likely those related to E6-AP substrates, may alter the developmental trajectory of neuronal structures including dendrites, spines and synaptic proteins, which leads to disrupted activity/experience-dependent synaptic plasticity and maturation. To test this hypothesis, we conducted a detailed investigation on neuronal morphology and electrophysiological properties in the prefrontal cortex (PFC) layer 5 (L5) corticostriatal pyramidal neurons (target neurons). We found smaller soma size in the maternal Ube3a deficient mice (m-/p+; 'AS' mice) at postnatal 17-19 (P17-19), P28-35 and older than 70 days (>P70), and decreased basal dendritic processes at P28-35. Surprisingly, both excitatory and inhibitory miniature postsynaptic currents (mEPSCs and mIPSCs) decreased on these neurons. These neurons also exhibited abnormalities in the local neural circuits, short-term synaptic plasticity and AMPA/NMDA ratio: the excitatory inputs from L2/3 and L5A, and inhibitory inputs from L5 significantly reduced in AS mice from P17-19; Both the release probability (Pr) and readily-releasable vesicle (RRV) pool replenishment of presynaptic neurons of the target neurons were disrupted at P17-19 and P28-35, and the change of RRV pool replenishment maintained through adulthood (>P70). The AMPA/NMDA ratio showed abnormality in the L5 corticostriatal neurons of PFC in AS mice older than P28-35, during which it decreased significantly compared to that of age-matched WT littermates. Western Blot analysis revealed that the expression level of a key regulator of the cytoskeleton system, Rho family small GTPase cell division control protein 42 homolog (cdc42), reduced significantly in the PFC of AS mice at P28-35.These impairments of synaptic transmission and short-term synaptic plasticity may account for the impaired neuronal morphology and synaptic deficits observed in the PFC target neurons, and contribute to the phenotypes in AS model mice. The present work reveals for the first time that the E6-AP deficiency influences brain function in both brain region-specific and age-dependent ways, demonstrates the functional impairment at the neural circuit level, and reveals that the presynaptic mechanisms are disrupted in AS model. These novel findings shed light on our understanding of the AS pathogenesis and inform potential novel therapeutic explorations.Dissertation/ThesisDoctoral Dissertation Neuroscience 201
Motor Learning Loss Due to MEK1 Hyperactivation in Cortical Excitatory Neurons
No full textabstract: Rasopathies are a family of developmental syndromes that exhibit craniofacial abnormalities, cognitive disabilities, developmental delay and increased risk of cancer. However, little is known about the pathogenesis of developmental defects in the nervous system. Frequently, gain-of-function mutations in the Ras/Raf/MEK/ERK cascade (aka ERK/MAPK) are associated with the observed pathogenesis. My research focuses on defining the relationship between increased ERK/MAPK signaling and its effects on the nervous system, specifically in the context of motor learning. Motor function depends on several neuroanatomically distinct regions, especially the spinal cord, cerebellum, striatum, and cerebral cortex. We tested whether hyperactivation of ERK/MAPK specifically in the cortex was sufficient to drive changes in motor function. We used a series of genetically modified mouse models and cre-lox technology to hyperactivate ERK/MAPK in the cerebral cortex. Nex:Cre/NeuroD6:Cre was employed to express a constitutively active MEK mutation throughout all layers of the cerebral cortex from an early stage of development. RBP4:Cre, caMEK only exhibited hyper activation in cortical glutamatergic neurons responsible for cortical output (neurons in layer V of the cerebral cortex). First, the two mouse strains were tested in an open field paradigm to assess global locomotor abilities and overall fitness for fine motor tasks. Next, a skilled motor reaching task was used to evaluate motor learning capabilities. The results show that Nex:Cre/NeuroD6:Cre, caMEK mutants do not learn the motor reaching task, although they performed normally on the open field task. Preliminary results suggest RBP4:Cre, caMEK mutants exhibit normal locomotor capabilities and a partial lack of learning. The difference in motor learning capabilities might be explained by the extent of altered connectivity in different regions of the corticospinal tract. Once we have identified the neuropathological effects of various layers in the cortex we will be able to determine whether therapeutic interventions are sufficient to reverse these learning defects
ERK/MAPK Requirements for the Development of Long-Range Axonal Projections and Motor Learning in Cortical Glutamatergic Neurons
No full textabstract: The RASopathies are a collection of developmental diseases caused by germline mutations in components of the RAS/MAPK signaling pathway and is one of the world’s most common set of genetic diseases. A majority of these mutations result in an upregulation of RAS/MAPK signaling and cause a variety of both physical and neurological symptoms. Neurodevelopmental symptoms of the RASopathies include cognitive and motor delays, learning and intellectual disabilities, and various behavioral problems. Recent noninvasive imaging studies have detected widespread abnormalities within white matter tracts in the brains of RASopathy patients. These abnormalities are believed to be indicative of underlying connectivity deficits and a possible source of the behavioral and cognitive deficits. To evaluate these long-range connectivity and behavioral issues in a cell-autonomous manner, MEK1 loss- and gain-of-function (LoF and GoF) mutations were induced solely in the cortical glutamatergic neurons using a Nex:Cre mouse model. Layer autonomous effects of the cortex were also tested in the GoF mouse using a layer 5 specific Rbp4:Cre mouse. Immunohistochemical analysis showed that activated ERK1/2 (P-ERK1/2) was expressed in high levels in the axonal compartments and reduced levels in the soma when compared to control mice. Axonal tract tracing using a lipophilic dye and an adeno-associated viral (AAV) tract tracing vector, identified significant corticospinal tract (CST) elongation deficits in the LoF and GoF Nex:Cre mouse and in the GoF Rbp4:Cre mouse. AAV tract tracing was further used to identify significant deficits in axonal innervation of the contralateral cortex, the dorsal striatum, and the hind brain of the Nex:Cre GoF mouse and the contralateral cortex and dorsal striatum of the Rbp4:Cre mouse. Behavioral testing of the Nex:Cre GoF mouse indicated deficits in motor learning acquisition while the Rbp4:Cre GoF mouse showed no failure to acquire motor skills as tested. Analysis of the expression levels of the immediate early gene ARC in Nex:Cre and Rbp4:Cre mice showed a specific reduction in a cell- and layer-autonomous manner. These findings suggest that hyperactivation of the RAS/MAPK pathway in cortical glutamatergic neurons, induces changes to the expression patterns of P-ERK1/2, disrupts axonal elongation and innervation patterns, and disrupts motor learning abilities.Dissertation/ThesisDoctoral Dissertation Molecular and Cellular Biology 201
