Ena/VASP proteins regulate vertebrate nervous system development : a thesis presented

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.Includes bibliographical references.Nervous system development is a complex morphogenetic process. Cell migration, axon guidance and many other regulated cell shape changes build a functional nervous system. These processes depend upon regulation of the actin cytoskeleton. Ena/VASP proteins are able to remodel the actin cytoskeleton in response to extracellular signals and have been shown to regulate the motility and morphology of a variety of cells. I have investigated the in vivo requirement for the vertebrate family members, Mena and VASP in nervous system development. I show that Mena and VASP are required for viability and the formation of the neural tube, spinal nerves and several brain commissures. Furthermore, I have investigated Ena/VASP function in neuronal cell migration using an in vitro assay and demonstrate that Ena/VASP proteins regulate the migration of cerebellar granule cells.by Annabelle Sheila Menzies.Ph.D

14-3-3 proteins stabilize LGI1-ADAM22 levels to regulate seizure thresholds in mice

新たなてんかん治療戦略を提案 --脳の過剰興奮を阻止するタンパク質ADAM22の量が鍵--. 京都大学プレスリリース. 2021-12-15.What percentage of the protein function is required to prevent disease symptoms is a fundamental question in genetic disorders. Decreased transsynaptic LGI1-ADAM22 protein complexes, because of their mutations or autoantibodies, cause epilepsy and amnesia. However, it remains unclear how LGI1-ADAM22 levels are regulated and how much LGI1-ADAM22 function is required. Here, by genetic and structural analysis, we demonstrate that quantitative dual phosphorylation of ADAM22 by protein kinase A (PKA) mediates high-affinity binding of ADAM22 to dimerized 14-3-3. This interaction protects LGI1-ADAM22 from endocytosis-dependent degradation. Accordingly, forskolin-induced PKA activation increases ADAM22 levels. Leveraging a series of ADAM22 and LGI1 hypomorphic mice, we find that ∼50% of LGI1 and ∼10% of ADAM22 levels are sufficient to prevent lethal epilepsy. Furthermore, ADAM22 function is required in excitatory and inhibitory neurons. These results suggest strategies to increase LGI1-ADAM22 complexes over the required levels by targeting PKA or 14-3-3 for epilepsy treatment

Disease-associated modulation of adult hippocampal neurogenesis

Adult neurogenesis has been the focus of over 1500 articles in the past 10 years. Evidence for the continuous production of new neurons in the adult brain has raised hopes for new therapeutic approaches. On the other hand, the generation of new neurons is modulated in several neurological diseases and disorders, suggesting the involvement of the adult neurogenesis in their pathogenesis. Therefore, a better understanding of the disease-associated modulation of adult neurogenesis is essential for determining the most effective therapeutic strategy. The purpose of this doctoral project was to investigate long-term adult hippocampal neurogenesis changes in two disease models. BrdU labeling in combination with various cellular markers, and genetic fate-mapping approach were used to reach this goal. In the first experiment, the impact of the BeAN strain of the Theiler’s virus on hippocampal cell proliferation and neuronal progenitors was evaluated in two mouse strains which differ in the disease course. It was shown that Theiler’s murine encephalomyelitis virus can exert delayed effects on the hippocampal neurogenesis with long-term changes evident 90 days following the infection. The hippocampal changes proved to depend on strain susceptibility and might have been affected by microglial cells. In the second experiment, hippocampal neurogenesis was analyzed based on genetic fate mapping of transgenic animals in the amygdala-kindling model of epilepsy. The number of new granule neurons added to the dentate gyrus was increased in kindled animals. A prior seizure history proved to be sufficient to induce a long-term net effect on neuron addition and an ongoing occurrence of seizures did not further increase the number of new neurons. Hypertrophic astrocytes were observed in the kindled animals suggesting that seizures result in structural changes of astrocytes that could be detected long after the termination of the insults. The results of the experiments indicated the importance of methodological considerations in chronic studies of neurogenesis

High resolution imaging to unveil the subcellular layout of the cannabinoid type-1 receptor in rodent models of brain disease

179 p.The present Thesis focuses on the description of new subcellular localizations of CB1 receptors in normal brain and the study of the CB1 receptor expression in certain pathophysiological states. Different histological techniques have been crucial in defining the CB1 receptor expression and localization at the cellular level. However, it is extremely difficult to identify the subcellular distribution of CB1 receptors in some cell-types due to its low expression level on those cells. Moreover, it remains a key question to know the pattern of the subcellular CB1 receptor expression and distribution under pathological states. The high resolution immunoelectron microscopy applied in this study has shown to be an excellent approach for the fine detection of CB1 receptors in the brain. In particular, the single pre-embedding immunogold method for electron microscopy based on the use of specific primary CB1 receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments was used, as well as the combined pre-embedding immunogold and immunoperoxidase method that implied the additional use of biotinylated secondary antibodies and avidin-biotin complex for the simultaneous localization of CB1 receptors and protein markers of specific brain cells or synapses

Alteration of inhibitory circuit and enhanced seizure propensity in oligophrenin-1 knock-out mice, a murine model of X-linked intellectual disability

Durante il tirocinio di tesi ho analizzato la suscettibilità alle crisi epilettiche e la circuiteria inibitoria dell’ippocampo funzionale in un modello murino di disabilità intellettiva, in particolare in topi knock out (KO) per il gene codificante l’oligofrenina-1 (OPHN-1). Con l’espressione “disabilità intellettiva” si indica un complesso disturbo del sistema nervoso centrale caratterizzato da deficit cognitivi spesso accompagnati da fenomeni epilettici e la cui patogenesi è ancora largamente sconosciuta. È stato tuttavia stabilito un contributo genetico e tra le possibili cause la più frequente è la forma X-linked, così definita in quanto legata a mutazioni di geni localizzati sul cromosoma X. Tra i geni convolti in questo tipo di patologia vi è l’oligofrenina-1 che codifica per una proteina appartenente alla famiglia delle Rho GTPasi activating protein (GAP).) Mutazioni a carico di questo gene sono state osservate in pazienti che mostravano anche l’insorgenza di fenomeni epilettici, ma la relazione tra disalbilità intellettiva e ipereccitabilità resta ancora da investigare. L’OPHN-1 viene espressa in maniera ubiquitaria sia durante lo sviluppo che in età adulta. A livello cellulare, OPHN-1 è espressa sia nelle cellule gliali che in quelle neuronali dove interagisce con l’actina svolgendo quindi un importante ruolo nei fenomeni di maturazione morfologica e migrazione cellulare (crescita assonale e sviluppo delle arborizzazioni dendritiche). Inoltre, OPHN-1 mostra una localizzazione sia pre che post sinaptica e risulta quindi essere coinvolta nella formazione delle sinapsi e nei processi di trasmissione sinaptica eccitatoria ed inibitoria. I topi OPHN-1 KO mostrano alterazioni nella memoria spaziale e nel comportamento sociale e presentano un fenotipo immaturo delle spine dendritiche dei neuroni piramidali dell’area CA1 dell’ippocampo associato ad un’alterata trasmissione sinaptica, sia eccitatoria che inibitoria. Per lo studio della circuiteria inibitoria dell’ippocampo, abbiamo inizialmente registrato l’attività spontanea dell’ippocampo in vivo (potenziali locali di campo) mediante l’impianto cronico di elettrodi bipolari. I potenziali di campo sono stati registrati un’ora al giorno, per 3 giorni consecutivi al termine dei quali sono stati analizzati prendendo in considerazione diversi parametri, quali la frequenza di crisi, il tempo speso in crisi, la durata media delle crisi e infine la frequenza delle punte interictali. Dai risultati si evidenzia una maggiore ipereccitabilità della circuiteria ippocampale dei topi KO rispetto ai wild type (WT), e soprattutto la presenza di crisi spontanee. Successivamente abbiamo effettuato esperimenti di immunoistochimica per analizzare diversi tipi di interneuroni GABAergici. In particolare, dalle conte stereologiche di interneuroni NPY, parvalbumina (PV) e somatostatina (SOM) positivi nella regione dell’ilo dell’ippocampo, è emerso che i topi OPHN-1 KO presentano un minor numero di cellule NPY-positive. Sono stati analizzati anche i bottoni sinaptici PV, evidenziando un aumento nei topi KO. Inoltre, per studiare la suscettibilità alle crisi, abbiamo utilizzato il modello dell’epilessia del lobo temporale indotta mediante somministrazione intraperitoneale di kainato (KA, agonista dei recettori kainato glutammatergici). I topi sono stati poi osservati per le due ore successive all’iniezione assegnando ogni dieci minuti un punteggio che varia da 0 a 7 in base al tipo di attività mostrata (0: attività normale, 4: crisi motorie limbiche, 7: morte) e anche in questo caso i KO sono risultati più suscettibili alle crisi rispetto ai WT. Due settimane dopo l’iniezione di KA, abbiamo poi analizzato i cambiamenti anatomici conseguenti alle crisi indotte e abbiamo messo in evidenza come la maggiore suscettibilità sia associata a un maggior danno nei KO, in termine di morte di interneuroni (PV, SOM e NPY) e sovra-espressione di NPY. I nostri risultati dimostrano che la perdita di funzione del gene oligofrenina-1 provoca alterazioni nella circuiteria inibitoria ippocampale e una suscettibilità alle crisi epilettiche

Disease-associated modulation of adult hippocampal neurogenesis

Adult neurogenesis has been the focus of over 1500 articles in the past 10 years. Evidence for the continuous production of new neurons in the adult brain has raised hopes for new therapeutic approaches. On the other hand, the generation of new neurons is modulated in several neurological diseases and disorders, suggesting the involvement of the adult neurogenesis in their pathogenesis. Therefore, a better understanding of the disease-associated modulation of adult neurogenesis is essential for determining the most effective therapeutic strategy. The purpose of this doctoral project was to investigate long-term adult hippocampal neurogenesis changes in two disease models. BrdU labeling in combination with various cellular markers, and genetic fate-mapping approach were used to reach this goal. In the first experiment, the impact of the BeAN strain of the Theiler’s virus on hippocampal cell proliferation and neuronal progenitors was evaluated in two mouse strains which differ in the disease course. It was shown that Theiler’s murine encephalomyelitis virus can exert delayed effects on the hippocampal neurogenesis with long-term changes evident 90 days following the infection. The hippocampal changes proved to depend on strain susceptibility and might have been affected by microglial cells. In the second experiment, hippocampal neurogenesis was analyzed based on genetic fate mapping of transgenic animals in the amygdala-kindling model of epilepsy. The number of new granule neurons added to the dentate gyrus was increased in kindled animals. A prior seizure history proved to be sufficient to induce a long-term net effect on neuron addition and an ongoing occurrence of seizures did not further increase the number of new neurons. Hypertrophic astrocytes were observed in the kindled animals suggesting that seizures result in structural changes of astrocytes that could be detected long after the termination of the insults. The results of the experiments indicated the importance of methodological considerations in chronic studies of neurogenesis