Microtubule-severing proteins and their role in the development and degeneration of the central nervous system

Neuronal cells are among the most morphologically elaborate cells in the human body. The creation, maintenance and modification of such high complexity are dependent on the organization of neuronal cytoskeleton elements. Microtubules play an essential role in this regard, thanks to their ability to form a resistant and yet plastic cytoskeleton. The acquisition of a more rigid or a more dynamic configuration depends upon the coordination of a large number of regulatory proteins. The present work is on a poorly characterized but translationally promising family of such regulatory proteins called microtubule-severing proteins. The work focuses on two such proteins in two different scenarios: fidgetin in neurodevelopment and spastin in neurodegeneration. Fidgetin was found to play a unique role in neurodevelopment by targeting the dynamic domain of the axonal microtubule as opposed to its stable domain, with fidgetin thus behaving as a microtubule plasticity suppressor. Its inhibition was found to boost the dynamic microtubule mass of the axon, thereby enhancing neuroplastic characteristics such as axon growth and the number of processes extended by an individual neuron. This profound effect on dynamic microtubules has interesting translational implications in neuropathology: from treating the injured central nervous system to better understanding neurodevelopmental disorders such as autism. On the other side of the spectrum, there is spastin, mutations of which cause a selective slow degeneration of first order neurons from the cortico-spinal tracts. The prevailing idea of this neurodegeneration is that neurotoxic effect of mutated spastin occurs through a loss-of-function mechanism. The present study challenges this concept by demonstrating the existence of an alternative gain-of-function mechanism of action. The data suggest that mutated spastin could induce neurodegeneration by aberrantly activating kinases, in particular casein kinase II, which in turn, compromises vital neuronal processes such as intracellular transport. Continuation of the work will open the door to a completely new translational paradigm to find an efficient treatment to this incurable condition.Ph.D., Neuroscience -- Drexel University, 201

Curcumin protects against NMDA-induced toxicity: A possible role for NR2A subunit

PURPOSE. Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDAinduced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). METHODS. NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca 2ϩ ] i . RESULTS. Curcumin dose-and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDAinduced [Ca 2ϩ ] i rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca 2ϩ ] i . The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. CONCLUSIONS. These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs. (Invest Ophthalmol Vis Sci

Use of induced pluripotent stem cell models to probe the pathogenesis of Choroideremia and to develop a potential treatment

Choroideremia (CHM) is a rare monogenic, X-linked recessive inherited retinal degeneration resulting from mutations in the Rab Escort Protein-1 (REP1) encoding CHM gene. The primary retinal cell type leading to CHM is unknown. In this study, we explored the utility of induced pluripotent stem cell-derived models of retinal pigmented epithelium (iPSC-RPE) to study disease pathogenesis and a potential gene-based intervention in four different genetically distinct forms of CHM. A number of abnormal cell biologic, biochemical, and physiologic functions were identified in the CHM mutant cells. We then identified a recombinant adeno-associated virus (AAV) serotype, AAV7m8, that is optimal for both delivering transgenes to iPSC-RPEs as well as to appropriate target cells (RPE cells and rod photoreceptors) in the primate retina. To establish the proof of concept of AAV7m8 mediated CHM gene therapy, we developed AAV7m8.hCHM, which delivers the human CHM cDNA under control of CMV-enhanced chicken β-actin promoter (CßA). Delivery of AAV7m8.hCHM to CHM iPSC-RPEs restored protein prenylation, trafficking and phagocytosis. The results confirm that AAV-mediated delivery of the REP1-encoding gene can rescue defects in CHM iPSC-RPE regardless of the type of disease-causing mutation. The results also extend our understanding of mechanisms involved in the pathophysiology of choroideremia. Keywords: Choroideremia, Human iPSCs, Retinal pigmented epithelium, Gene therapy, Adeno-associated virus, Phagocytosis, Prenylation, REP

Mutant spastin proteins promote deficits in axonal transport through an isoform-specific mechanism involving casein kinase 2 activation

Mutations of various genes cause hereditary spastic paraplegia (HSP), a neurological disease involving dying-back degeneration of upper motor neurons. From these, mutations in the SPAST gene encoding the microtubule-severing protein spastin account for most HSP cases. Cumulative genetic and experimental evidence suggests that alterations in various intracellular trafficking events, including fast axonal transport (FAT), may contribute to HSP pathogenesis. However, the mechanisms linking SPAST mutations to such deficits remain largely unknown. Experiments presented here using isolated squid axoplasm reveal inhibition of FAT as a common toxic effect elicited by spastin proteins with different HSP mutations, independent of microtubule-binding or severing activity. Mutant spastin proteins produce this toxic effect only when presented as the tissue-specific M1 isoform, not when presented as the ubiquitously-expressed shorter M87 isoform. Biochemical and pharmacological experiments further indicate that the toxic effects of mutant M1 spastins on FAT involve casein kinase 2 (CK2) activation. In mammalian cells, expression of mutant M1 spastins, but not their mutant M87 counterparts, promotes abnormalities in the distribution of intracellular organelles that are correctable by pharmacological CK2 inhibition. Collectively, these results demonstrate isoform-specific toxic effects of mutant M1 spastin on FAT, and identify CK2 as a critical mediator of these effects.Fil: Leo, Lanfranco. Drexel University College of Medicine; Estados UnidosFil: Weissmann, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Chicago; Estados UnidosFil: Burns, Matthew. University of Chicago; Estados UnidosFil: Kang, Minsu. University of Chicago; Estados Unidos. Marine Biological Laboratory; Estados UnidosFil: Song, Yuyu. Marine Biological Laboratory; Estados Unidos. Yale University; Estados UnidosFil: Qiang, Liang. Drexel University College of Medicine; Estados UnidosFil: Brady, Scott T.. University of Chicago; Estados Unidos. Marine Biological Laboratory; Estados UnidosFil: Baas, Peter W.. Drexel University College of Medicine; Estados UnidosFil: Morfini, Gerardo. University of Chicago; Estados Unidos. Marine Biological Laboratory; Estados Unido

Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics

Vertebrate Fidgetin Restrains Axonal Growth by Severing Labile Domains of Microtubules

Individual microtubules (MTs) in the axon consist of a stable domain that is highly acetylated and a labile domain that is not. Traditional MT-severing proteins preferentially cut the MT in the stable domain. In Drosophila, fidgetin behaves in this fashion, with targeted knockdown resulting in neurons with a higher fraction of acetylated (stable) MT mass in their axons. Conversely, in a fidgetin knockout mouse, the fraction of MT mass that is acetylated is lower than in the control animal. When fidgetin is depleted from cultured rodent neurons, there is a 62% increase in axonal MT mass, all of which is labile. Concomitantly, there are more minor processes and a longer axon. Together with experimental data showing that vertebrate fidgetin targets unacetylated tubulin, these results indicate that vertebrate fidgetin (unlike its fly ortholog) regulates neuronal development by tamping back the expansion of the labile domains of MTs

La Padania, una regione italiana in Europa

Dettagliata analisi della area padana e delle sue caratteristiche con approfondimenti sulla struttura ed evoluzione della sua economia, delle sue risorse geo-economiche e infrastrutturali con riferimento anche alla collocazione della Padania del più ampio contesto europeo e dei mercati internazioali.- Indice #6- Introduzione, Marcello Pacini #10- Prima parte Le caratteristiche dell’area #23- Struttura ed evoluzione dell’economia padana, Alberto Bramanti e Lanfranco Senn #24- Internazionalizzazione dell’economia padana, Sergio Alessandrini #130- Il sistema dei trasporti padano: progetti e prospettive, Centro Studi sui Sistemi di Trasporto #204- Competere in prospettiva europea: la Padania nel quadro delle grandi regioni economiche d’Europa, Roberto P. Camagni #294- Pieno e vuoto. La risorsa spazio in Padania, Luigi Mazza #310- Acque e rifiuti. Da risorse a fattori limitanti lo sviluppo nel bacino del Po, Pier Francesco Ghetti #332- Seconda parte Il contesto europeo #354- Parigi-Lione dieci anni fa. Torino-Lione tra dieci anni?, Olivier Klein #356- Reti ad alta velocità e territori economici in Europa, Olivier Klein #372- Randstad Holland: sviluppo e pianificazione di una metropoli complessivamente concorrenziale, Ilaria Bramezza e Leo van den Berg #394- La regione Reno-Ruhr: la metropoli flessibile, Klaus R. Kunzmann #44