Paradoxical signaling regulates structural plasticity in dendritic spines

Transient spine enlargement (3-5 min timescale) is an important event associated with the structural plasticity of dendritic spines. Many of the molecular mechanisms associated with transient spine enlargement have been identified experimentally. Here, we use a systems biology approach to construct a mathematical model of biochemical signaling and actin-mediated transient spine expansion in response to calcium-influx due to NMDA receptor activation. We have identified that a key feature of this signaling network is the paradoxical signaling loop. Paradoxical components act bifunctionally in signaling networks and their role is to control both the activation and inhibition of a desired response function (protein activity or spine volume). Using ordinary differential equation (ODE)-based modeling, we show that the dynamics of different regulators of transient spine expansion including CaMKII, RhoA, and Cdc42 and the spine volume can be described using paradoxical signaling loops. Our model is able to capture the experimentally observed dynamics of transient spine volume. Furthermore, we show that actin remodeling events provide a robustness to spine volume dynamics. We also generate experimentally testable predictions about the role of different components and parameters of the network on spine dynamics

The role of Cdc42 GTPase during synaptic cooperation

Tese de mestrado, Neurociências, Universidade de Lisboa, Faculdade de Medicina, 2019A memória refere-se ao armazenamento de informação previamente aprendida. Esta capacidade permite que os animais ajustem e adaptem o seu comportamento ao ambiente, de acordo com aquilo que experienciaram. Assim, um dos mais fascinantes e centrais desafios da ciência moderna é a compreensão dos mecanismos neuronais que estão na base da aquisição e armazenamento de informação. Atualmente, pensa-se que a plasticidade sináptica esteja envolvida numa grande variedade de funções cerebrais, incluindo aprendizagem e memória. Em particular, a investigação desenvolvida tem-se concentrado em plasticidade sináptica dependente de atividade neuronal, como é o exemplo da potenciação de longa duração (LTP, do inglês long-term potentiation) ou da depressão de longa-duração (LTD, do inglês long-term depression) enquanto modelos celulares que estão na base da memória. Modelos clássicos de manutenção de LTP distinguem, pelo menos, duas fases: uma fase inicial (E-LTP, do inglês early long-term potentiation), independente de síntese proteica, que persiste durante minutos e uma fase tardia (L-LTP, do inglês late long-term potentiation), dependente de síntese proteica. A indução de LTP é específica em relação ao input, ou seja, apenas as sinapses ativadas são potenciadas. Isto implica que macromoléculas necessárias para a manutenção de LTP, PRPs (do inglês plasticity-related proteins), devem ser, de alguma forma, recrutadas para sinapses ativadas. A hipótese de tagging e captura sináptica (STC, do inglês Synaptic Tagging and Capture), introduzida por Frey e Morris em 1997, propôs um mecanismo celular que concilia a especificidade de input da plasticidade sináptica com a alocação de PRPs. O modelo STC propõe que a atividade neuronal leva à formação de uma tag nas sinapses ativadas. A tag, temporalmente e espacialmente limitada, permite a essas sinapses a captura de moléculas necessárias à manutenção da plasticidade sináptica. Assim, a indução de LTP através de tetanização forte tem como consequência dois eventos dissociáveis: o estabelecimento local de uma tag sináptica e a síntese de PRPs. A indução de LTP através de tetanização fraca não leva à síntese de PRPs; no entanto, leva ao estabelecimento de uma “tag”. De uma forma geral, é a interação entre a tag e as PRPs que permite a manutenção da plasticidade sináptica. Neste contexto, podem surgir formas cooperativas de plasticidade sináptica, como observado em experiências anteriores: formas transientes de plasticidade, induzidas por uma estimulação fraca das sinapses, podem ser convertidas em formas persistentes através da utilização de PRPs sintetizadas devido à estimulação forte recebida por outro grupo de sinapses. A competição sináptica é outra possibilidade, em situações de menor disponibilidade de PRPs ou maior número de tags ativas, por exemplo. Embora não sejam ainda totalmente compreendidas, as vias de transdução de sinal responsáveis pelo estabelecimento da tag começam a ser elucidadas. De acordo com aquilo que se sabe atualmente, a tag dificilmente será equivalente a uma molécula (ou a um pequeno conjunto de moléculas). Em vez disso, a tag deve ser vista enquanto uma alteração local e transiente do estado da sinapse que, muito provavelmente, envolve uma complexa rede de proteínas e interações. Resultados anteriores do nosso grupo de laboratório demonstraram que o citoesqueleto de actina desempenha um papel crucial na captura de PRPs tanto para LTP como para LTD, suportando a hipótese de que uma remodelação dos filamentos de actina (F-actin), dependente de atividade sináptica, torna a sinapse localmente e transientemente permissiva a modificações plásticas. A proteína quinase CaMKII (do inglês Ca2+/calmodulin-dependent protein kinase II) demonstrou ser necessária para mecanismos de captura sináptica. A indução de plasticidade sináptica leva à dissociação da subunidade β da CaMKII do citoesqueleto de actina, algo necessário para a remodelação da sinapse. Esta deslocação (de cerca de 1 minuto) gera um sinal local que pode estar na base da tag sináptica. Curiosamente, a CaMKII leva à ativação de Cdc42 (do inglês cell division control protein 42 homolog), uma proteína pertencente à família das Rho GTPases, que também desempenha um papel central na regulação do citoesqueleto de actina nas espinhas dendríticas. A cofilina é uma proteína de distribuição ubíqua, cuja atividade leva à despolimerização dos filamentos de actina. Sabe-se que a Cdc42 está acima de PAK (do inglês p21-activated kinase), que promove a inibição da cofilina de duas formas. Por um lado, PAK fosforila e ativa a quinase LIMK (LIM-kinase), que, por sua vez, fosforila cofilina; por outro, bloqueia a atividade da fosfatase SSH (do inglês Slingshot homologue), inibindo assim a desfosforilação da cofilina e, consequentemente, a sua atividade. A Cdc42 é também responsável pela interação com a via N-WASP que leva à ativação do complexo Arp2/3, conhecido pelo seu papel na polimerização do citoesqueleto de actina. Foi previamente demonstrado que, ao contrário de outras Rho GTPases, a ativação da Cdc42 é restrita às espinhas dendríticas estimuladas (ou seja, demonstra especificidade de input), persiste durante mais de 30 minutos e depende da sinalização de BDNF (do inglês brain-derived neurotrophic factor). Aqui, colocamos a hipótese de que a Cdc42 desempenha um papel essencial na persistência da plasticidade sináptica, sendo necessária para o estabelecimento de uma tag sináptica. Para testar esta hipótese, administrámos ML141, um inibidor farmacológico reversível e altamente seletivo para a Cdc42, em fatias hipocampais de ratos juvenis em simultâneo com o registo eletrofisiológico de fEPSPs (do inglês field excitatory post-synaptic potentials) no stratum radiatum da área CA1. Em condições de controlo, uma tetanização forte ao nível das colaterais de Schaffer leva à indução de uma forma persistente de LTP. Contudo, demonstrámos que, se o mesmo tipo de tetanização coincidir temporalmente com o período de aplicação do inibidor de Cdc42, a potenciação decai rapidamente para valores de baseline, como se observa em formas transientes de LTP. Estes resultados sugerem que a indução de formas persistentes de LTP depende da atividade de Cdc42. Para além de se ter revelado enquanto necessária para a indução de LTP persistente, a Cdc42 revelou também ser necessária para a sua manutenção de acordo com uma janela temporal limitada. Ou seja, quando a aplicação do inibidor de Cdc42 é feita 40 a 70 minutos depois da indução de LTP, podemos observar um decaimento da potenciação sináptica. No entanto, a destabilização da manutenção de LTP persistente deixa de se verificar quando a aplicação do inibidor é feita 70 a 100 minutos depois da indução de plasticidade sináptica. Finalmente, foram efetuadas experiências de cooperação sináptica. Primeiro, uma via é estimulada com tetanização fraca; passados 30 minutos, uma segunda via independente é estimulada com tetanização forte, que induz uma forma persistente de plasticidade. Em condições de controlo, a via que recebeu a estimulação fraca – que, por si só, levaria à expressão de uma forma transiente de LTP – é capaz de converter o seu LTP numa forma de LTP mais estável e persistente devido à estimulação forte na outra via. No entanto, quando a atividade de Cdc42 é inibida entre as duas estimulações, apenas a via que é estimulada com tetanização forte expressa uma forma persistente de LTP. Estes resultados sugerem que a inibição de Cdc42 interferiu com mecanismos de tagging e captura de macromoléculas necessárias à manutenção da plasticidade sináptica. De uma forma geral, os nossos resultados apoiam a hipótese de que a Cdc42, ao regular o citoesqueleto de actina, interfere com a plasticidade sináptica, desempenhando um papel crucial na mesma. Estas e outras observações relativas aos mecanismos através dos quais o citoesqueleto é remodelado em consequência de atividade neuronal podem ter profundas implicações na compreensão dos mecanismos que estão subjacentes aos processos de memória e de aprendizagem. Para além disso, podem fornecer importantes alvos terapêuticos para doenças neuropsiquiátricas (como doença de Alzheimer e esquizofrenia, por exemplo) em que foram já identificadas disfunções relacionadas com a actina ou com a complexa rede dos seus reguladores e interações que estabelecem entre si.Maintained forms of Long-term potentiation (LTP) require de novo protein synthesis of plasticity-related proteins (PRPs). Since LTP is input-specific, it was proposed that activated synapses are tagged so that synthesized proteins are captured at modified synapses (synaptic tagging and capture hypothesis - STC). Although the nature of the synaptic tag remains unclear, it is generally accepted that it must be a local and transient molecular alteration caused by synaptic activation which can capture PRPs. Several molecules have been implicated in LTP maintenance by synaptic tagging and capture mechanisms, namely CaMKII, PKA and BDNF. Previous results from our laboratory group have shown a critical role of actin dynamics in the tagging and capture of PRPs in both LTP and LTD, supporting the hypothesis that an activity-dependent remodeling of F-actin through CaMKII activation, renders the synapse locally and transiently permissive to plasticity modifications. Interestingly, CaMKII leads to activation of Cdc42, a Rho GTPase that also plays a role in regulating the actin cytoskeleton in dendritic spines. Cdc42 is known to be upstream of PAK (p21-activated kinase), that phosphorylates and activates LIM-kinase (LIMK), which, in turn, phosphorylates cofilin, inhibiting its actin-depolymerizing activity. Cdc42 is also responsible for interacting with the WAVE1/N-WASP pathway to activate Arp2/3 complex-dependent actin polymerization. One hypothesis is that Cdc42 activity promotes actin polymerization. Since Cdc42 activity has been shown to be heavily restricted to the stimulated spine (input-specificity), last more than 30 minutes and is dependent on BDNF signaling, we hypothesize that Cdc42 plays a crucial role in the setting of the synaptic tag. Here, we assess the role of Cdc42 in synaptic tagging and LTP maintenance by pharmacologically inhibiting Cdc42 activity while performing electrophysiological recordings in rat hippocampal slices. We found that inhibition of Cdc42 does not interfere with the expression of a transient form of LTP but blocks the induction of a maintained form of LTP. Moreover, the inhibition of Cdc42 blocks the maintenance of synaptic plasticity within a limited time window. Finally, we show that Cdc42 inhibition interferes with synaptic tagging and capture mechanisms. Since Cdc42 activation promotes actin polymerization we propose that Cdc42 inhibition may interfere with the maintenance of LTP by promoting actin depolymerization. Our results suggest that, by interfering with the actin cytoskeleton, Cdc42 interferes with synaptic plasticity

Control of Neuronal Circuit Assembly by Gtpase Regulators

One of the most remarkable features of the central nervous system is the exquisite specificity of its synaptic connections, which is crucial for the functioning of neuronal circuits. Thus, understanding the cellular and molecular mechanisms leading to the precise assembly of neuronal circuits is a major focus of developmental neurobiology. The structural organization and specific connectivity of neuronal circuits arises from a series of morphological transformations: neuronal differentiation, migration, axonal guidance, axonal and dendritic arbor growth and, eventually, synapse formation. Changes in neuronal morphology are driven by cell intrinsic programs and by instructive signals from the environment, which are transduced by transmembrane receptors on the neuronal cell surface. Intracellularly, cytoskeletal rearrangements orchestrate the dynamic modification of neuronal morphology. A central question is how the activation of a neuronal cell surface receptor triggers the intracellular cytoskeletal rearrangements that mediate morphological transformations. A group of proteins linked to the regulation of cytoskeletal dynamics are the small GTPases of the Rho family. Small RhoGTPases are regulated by GTPase exchange factors (GEF) and GTPase activating proteins (GAP), which can switch GTPases into "on or off" states, respectively. It is thought that GEFs and GAPs function as intracellular mediators between transmembrane receptors and RhoGTPases to regulate cytoskeletal rearrangements. During my dissertation I identified the GAP α2-chimaerin as an essential downstream effector of the axon guidance receptor EphA4, in the assembly of neuronal locomotor circuits in the mouse. Furthermore, I identified two novel neuronal GAPs, mSYD-1A and mSYD-1B, which interact with components of the presynaptic active zone and which may contribute to presynaptic assembly downstream of synaptic cell surface receptors

Studies on the characterization of neuronal βPix isoform knockout mouse and the role of βPix-d in microtubule stabilization and neurite outgrowth

학위논문(박사)--서울대학교 대학원 :자연과학대학 생명과학부,2019. 8. 박동은.βPix activates Rho family small GTPases, Rac1 and Cdc42 as a guanine nucleotide exchange factor. Although overexpression of βPix in cultured neurons indicates that βPix is involved in spine morphogenesis and synapse formation in vitro, the in vivo role of βPix in neuron is not well understood. Recently, we generated βPix knockout mice that showed lethality at embryonic day 9.5. Here, we investigate the neuronal role of βPix using βPix heterozygous mice that are viable and fertile. βPix heterozygous mice show decreased expression level of βPix proteins in various tissues including brain. Cultured hippocampal neurons from βPix heterozygous mice show a decrease in neurite length and complexity as well as in synaptic density. Both excitatory and inhibitory synapse densities are decreased in these neurons. Golgi-staining of hippocampal tissues from the brain of these mice show reduced dendritic complexity and spine density in the hippocampal neurons. Expression levels of NMDA- and AMPA- receptor subunits and Git1 protein in hippocampal tissues are also decreased in these mice. Behaviorally, βPix heterozygous mice exhibit impaired social interaction. Altogether, these results indicate that βPix is required for neurite morphogenesis and synapse formation, and the reduced expression of βPix proteins results in a defect in social behavior. Among alternatively spliced βPix isoforms, βPix-b and βPix-d are expressed specifically in neurons, however their neuronal functions are poorly understood. Here, we generated neuronal βPix isoform knockout (KO) mice in which expression of the neuronal isoforms, βPix-b and βPix-d, are deleted. Loss of the neuronal βPix isoforms leads to reduced Rac1 and Cdc42 activities, decreased dendritic complexity and spine density, and increased GluN2B and CaMKIIα expression in the hippocampus in vivo. The defects in neurite development and dendritic spine maturation in cultured neuronal βPix isoform KO hippocampal neurons are recovered by expression of βPix-b or βPix-d. Furthermore, the neuronal βPix isoform KO neurons display decreased excitatory and inhibitory synaptic densities, which are rescued by expression of βPix-b or βPix-d. Neuronal βPix isoform KO mice have defects in long-term potentiation and showed impaired novel object recognition, startle response and prepulse inhibition, and low anxiety levels. Taken together, our research highlights that neuronal βPix isoforms are required for normal development of neuronal structures, synapse formation and behaviors in hippocampus. Microtubules are one of the major cytoskeletal components of neurites including axons and dendrites. The regulation of microtubule stability is important for appropriate neurite morphogenesis during neuronal development, which in turn is critical for establishment of functional neuronal connections. In neurons, βPix-b plays a role in dendritic spine morphogenesis that mainly involves reorganizations of actin filaments, but the neuronal role of βPix-d has not been revealed. In addition, the role of neuronal βPix isoforms during neurite outgrowth is not well understood. Here, we unveil a novel mechanism for βPix-d to regulate microtubule stabilization and neurite outgrowth. At DIV4, hippocampal neurons cultured from neuronal βPix isoform KO mice show defects in neurite length and complexity, and microtubule stability. Treatment of taxol that stabilizes microtubules alleviates the impairment of neurite morphology and microtubule stability in the neuronal βPix isoform KO neurons. Neuronal βPix isoforms increase microtubule stability and βPix-d stabilizes microtubules more than βPix-b. We also find the localization of βPix-d on microtubules. In addition, the neuronal βPix isoform KO neurons have a deficit in phosphorylation level of Stathmin1, a microtubule severing protein, at Ser16. Neuronal βPix isoforms recover the impairments of neurite morphology and phosphorylation level in Stathmin1 shown in the neuronal βPix isoform KO neurons and the levels of those recoveries are greater in expression of βPix-d than βPix-b. Furthermore, those recoveries by βPix-d in the neuronal βPix isoform KO neurons are not observed by inhibition of PAK1 activity that phosphorylates Stathmin1 at Ser16. Taken together, our present study show that βPix-d stabilizes microtubules and phosphorylates Stathmin1 at Ser16 with PAK1 more than βPix-b, and is consequently involved in neurite morphogenesis.βPix(beta-PAK (p21-activated kinase) interacting exchange factor)는 액틴 골격근(actin filament)과 미세소관(microutbule)의 핵심 조절 인자인 Rac1과 Cdc42 small GTPase를 활성화시키는 GEF(guanine nucleotide exchange factor)로 잘 알려져 있다. 신경세포에서 βPix는 축삭돌기(axon), 수상돌기가시(dendritic spine), 시냅스(synape) 형성을 조절한다고 알려져 있다. 본 연구에서는 지금까지 신경세포의 발달과정에 관여하는 βPix의 기능 연구가 in vitro에서 진행되었다는 점에 착안하여, 본 연구실에서 제작한 βPix heterozygous mouse와 신경세포 특이적 βPix isoform knockout mouse를 통해 신경세포 구조 발달과정에서 βPix의 in vivo 기능을 밝히고, 지금까지 알려지지 않은 신경세포 특이적 βPix isoform인 βPix-d의 기능을 밝히고자 하였다. βPix heterozygous mice는 뇌를 포함한 다양한 조직에서 βPix의 발현이 반으로 감소되어 있다. βPix heterozygous mice로부터 배양한 해마 신경세포는 신경돌기(neurite)의 길이가 짧고 복잡성이 단순화 되어 있으며 수상돌기가시(dendritic spine)의 밀도도 감소 되어있었다. 나아가 골지 염색한 뇌의 신경세포에서 수상돌기(dendrite) 가지의 복잡성과 수상돌기가시(dendritic spine)의 밀도에 결함이 있음을 in vivo에서도 확인하였다. 또한, 12주령의 βPix heterozygous mice의 해마에 시냅스 기능을 위해 필요한 NMDA 수용체와 AMPA 수용체의 subunit들의 발현이 감소되어 있고 βPix의 강력한 결합 단백질이자 βPix와 함께 수상돌기가시(dendritic spine) 발달을 조절한다고 알려진 Git1의 발현도 감소되어 있음을 관찰하였다. 종합하면, βPix가 신경돌기(neurite)와 수상돌기가시(dendritic spine)의 발달과정에 필수적임을 in vitro와 in vivo에서 확인하였다. βPix는 선택적 스플라이싱에 의해 신경세포 특이적으로 βPix-b와 βPix-d isoform을 발현하고 있고, 이러한 신경세포 특이적 βPix isoform에 대한 in vivo 기능은 보고된 바 없다. 본 연구실에서 제작한 신경세포 특이적 βPix isoform knockout mice의 뇌에는 신경세포 특이적 βPix isoform의 발현이 감소되어 있고, 특히 해마 내 Rac1과 Cdc42의 활성이 감소되어 있다. 신경세포 특이적 βPix isoform knockout mice의 in vivo 특징을 규명하기 위한 골지 염색에서 해마 신경세포 수상돌기(dendrite)의 복잡성과 수상돌기가시(dendritic spine)의 밀도에 결함이 관찰되고, 특히 사춘기 단계인 5주령 쥐의 해마 조직에서 NMDA 수용체 subunit 중 GluN2B와 CaMKIIα의 발현이 일시적으로 증가되어 있음을 확인하였다. 또한 신경세포 특이적 βPix isoform knockout mice의 배양한 해마 신경세포의 신경돌기(neurite)와 수상돌기가시(dendritic spine) 구조 발달에 결함이 있음을 확인하였고 더 나아가 흥분성 시냅스와 억제성 시냅스의 밀도 및 이러한 시냅스를 이루는 시냅스 후 구조와 시내스 전 구조의 크기가 감소되어 있음을 관찰하였다. 신경세포 특이적 βPix isoform knockout mice에서 배양한 해마 신경세포에 신경세포 특이적 βPix-b와 βPix-d를 각각 발현시켰을 때, 결함을 보였던 신경돌기(neurite)와 수상돌기가시(dendritic spine)의 발달, 흥분성 시냅스와 억제성 시냅스의 형성이 회복되었다. 흥미롭게도, βPix-b가 수상돌기가시(dendritic spine)와 흥분성 시냅스 발달에 βPix-d보다 더 필수적으로 작용하고, βPix-d는 신경돌기(neurite)와 억제성 시냅스 발달에 βPix-b보다 더 필수적으로 작용하는 것을 확인함으로써 신경세포 특이적 βPix isoform들간의 신경세포 내 차별화된 기능을 규명하였다. 신경돌기(neurite) 내 미세소관(microtubule) 안정화는 정상적인 신경돌기(neurite) 발달에 중요하고 이는 정상적인 신경망 형성에 필수적이다. 신경세포 특이적 βPix isoform knockout mice의 해마 신경세포는 발달과정 중 이른 시기에 신경돌기(neurite)의 길이와 복잡성에 결함을 보이고 신경돌기(neurite)를 구성하는 미세소관(microtubule)의 안정성도 떨어져 있음이 관찰되었다. 이들 신경세포에서 감소된 미세소관(microtubule)의 안전성을 taxol을 통해 회복시켰을 때 손상된 신경돌기(neurite)의 구조가 회복되는 것을 확인하였다. 또한, 신경세포 특이적 βPix isoform인 βPix-b와 βPix-d의 발현에 의해 미세소관(microtubule)의 안정성이 증가하였고 βPix-b보다 βPix-d에 의해 안정성이 더 크게 증가하였다. βPix-d의 세포 내 발현 위치는 미세소관(microtubule)과 일치하는 것을 확인하였다. 추가적으로 신경세포 특이적 βPix isoform knockout mice의 해마 신경세포에는 미세소관(microtubule)을 불안정화 시키는 단백질인 Stathmin1의 인산화가 감소되어 있음을 확인하였고, βPix-d에 의해 감소되었던 Stathmin1의 인산화가 회복됨도 확인하였다. 또한 같은 시기의 해마 신경세포의 구조도 신경세포 특이적 βPix isoform knockout mice에 βPix-d를 발현시키면 결함을 보였던 신경돌기(neurite)의 구조가 회복되는 것을 확인함으로써, βPix-d에 의해 Stathmin1의 활성이 감소되어 미세소관(microtubule)의 안정화가 증가됨으로써 신경돌기(neurite) 구조가 발달되는 것을 확인하였다. βPix의 결합 단백질인 PAK1이 Stathmin1을 직접적으로 인산화를 시켜준다는 보고가 있다. 신경세포 특이적 βPix isoform knockout mice의 해마 신경세포에서 PID(PAK inhibitory domain)의 발현에 의해 PAK1의 활성을 저해시키면, βPix-d를 발현하더라도 Stathmin1의 인산화 증가가 관찰되지 않았다. 이 결과는 βPix-d에 의한 Stathmin1의 인산화가 PAK1의 활성을 통한 것임을 제시한다. 본 연구는 βPix-d가 PAK1을 활성화시켜 Stathmin1의 인산화를 증가시킴으로써 미세소관을 안정화시켜 신경돌기 발달을 조절하는 신호전달 기전을 제시한다.ABSTRACT I TABLE OF CONTENTS IV LIST OF FIGURES VII BACKGROUND XI 1. βPix XI 2. Neuronal development XV 3. Neuronal structures and brain disorders XVIII 4. Microtubule stabilization in neurites XXI 5. Stathmin1 XXIV MATERIALS AND METHODS XXVII 1. Mice XXVII 2. Reagents and antibodies XXXIII 3. Western blot analysis XXXIV 4. Nissl staining XXXV 5. Primary hippocampal neuron culture and transfection XXXV 6. Immunocytochemistry XXXVI 7. Analysis of neuronal morphology and synapse density in cultured hippocampal neurons XXXVII 8. Golgi staining XXXVIII 9. Sholl analysis and analysis of spine density in Golgi stained tissues XXXVIII 10. In vivo glutathione S-transferase (GST) pull-down assay XXXVIII 11. Postsynaptic density (PSD) preparation from mouse brain XXXIX 12. Immunohistochemistry XL 13. Constructs XLI 14. Statistics XLI CHAPTER I. Studies on the characterization of βPix heterozygous mouse and neuronal βPix isoform knockout mouse 1 Abstract 2 Introduction 4 Results 7 1. Characterization of βPix heterozygous mouse 7 1.1. βPix heterozygous mice have decreased βPix expression level in brain. 7 1.2. Cultured hippocampal neurons from βPix heterozygous mice show defects in the development of neurite, dendritic spine and synapse. 7 1.3. Dendritic complexity and spine density are decreased in hippocampal neurons of βPix heterozygous mice in vivo. 12 1.4. Adult βPix heterozygous mice have decreased protein levels of glutamate receptor subunits and Git1. 19 1.5. βPix heterozygous mice have deficits in social interaction. 26 2. Characterization of neuronal βPix isoform knockout mouse 29 2.1. Neuronal βPix isoform KO mice display loss of βPix-b and βPix-d in brain. 29 2.2. Active Rac1 and Cdc42 are reduced in hippocampus of neuronal βPix isoform KO mice. 32 2.3. Dendritic complexity and spine density are decreased in hippocampal pyramidal neurons of neuronal βPix isoform KO mice. 32 2.4. Juvenile KO mice have increased levels of GluN2B and CaMKIIα expression. 42 2.5. βPix-b and βPix-d are required for growth cone development. 45 2.6. βPix-b and βPix-d are required for neurite development. 50 2.7. βPix-b and βPix-d are required for dendritic spine development. 55 2.8. βPix-b and βPix-d are required for synaptic development. 63 2.9. Neuronal βPix isoform KO mice have reduced LTP at hippocampal Schaffer collateral-CA1 synapses. 75 2.10. Neuronal βPix isoform KO mice show impaired recognition memory and startle response, and low anxiety levels. 80 Discussion 89 CHAPTER II. Studies on the role of βPix-d in microtubule stabilization and neurite outgrowth 100 Abstract 101 Introduction 103 Results 106 1. Neurite length and branching are decreased in hippocampal neurons from neuronal βPix isoform KO mice at DIV4. 106 2. Microtubule stability is decreased in the longest neurite extending from hippocampal neurons from neuronal βPix isoform KO mice. 111 3. Recovery of microtubule stability by taxol rescues the impaired neurite morphology in hippocampal neurons from neuronal βPix isoform KO mice. 114 4. βPix-d is required for microtubule stabilization. 117 5. βPix-d is co-localized with microtubule. 124 6. βPix-d is required for phosphorylation of Stathmin1 at Ser16 and neurite outgrowth. 134 7. βPix-d is required for PAK1-induced phosphorylation of Stathmin1 at Ser16 and neurite outgrowth. 142 Discussion 150 REFERENCES 158 국문초록 178Docto

ARHGAP22 DISRUPTION AFFECTS RAC1 SIGNALING PATHWAY AND RESULTS IN ALTERED FORMATION AND FUNCTION OF GLUTAMATERGIC SYNAPSES IN MOUSE HIPPOCAMPUS

The regulation of actin cytoskeleton operated by RhoGTPases is crucial for neuronal morphogenesis, especially for neurite elongation and branching, synaptogenesis and synaptic plasticity. Dysregulation of RhoGTPases leads to neuronal dysfunctions including intellectual disability, schizofrenia and Alzheimer disease. Rac1 is a member of the RhoGTPase family and it has been demonstrated to positively regulate dendritogenesis and dendritic spines formation and maturation. As well as other RhoGTPases, Rac1 activity is regulated principally by two kinds of molecules: GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins). Arhgap22 protein is a specific Rac1-Gap that promotes the inactivation of Rac1. Although it was previously reported that Arhgap22 transcripts is present in murine brain, its functions in neurons have not been studied yet. Here, we reported that Arhgap22 is expressed in mouse brain in a precise spatio-temporal window. Moreover, taking advantage of an animal mouse model knock out (KO) for Arhgap22, we described the effects of its silencing in hippocampal neurons. In vivo, Arhgap22 disruption led to an increase level of activated Rac1 and its downstream pathways, with a subsequent increase in dendritic spine density in CA1 region of hippocampus. Additionally, Arhgap22 lacking mice presented reduced AMPA receptors in the post-synaptic density of excitatory synapses and this alteration was reflected by the impairment in the induction and mainteinance of long-term potentiation (LTP). Arhgap22 KO mice presented also defects in cognitive tasks and decreased anxiety-like behaviours. In a nutshell, the results of this work suggested that Arhgap22 is a key regulator of Rac1 signaling and that affects the maturation of excitatory synapses, synaptic plasticity and cognitive functions

Angelman syndrome: advancing the research frontier of neurodevelopmental disorders

This report is a meeting summary of the 2010 Angelman Syndrome Foundation's scientific symposium on the neuroscience of UBE3A. Angelman syndrome is characterized by loss of speech, severe developmental delay, seizures, and ataxia. These core symptoms are caused by maternal allele disruptions of a single gene—UBE3A. UBE3A encodes an E3 ubiquitin ligase that targets certain proteins for proteasomal degradation. This biology has led to the expectation that the identification of Ube3a protein targets will lead to therapies for Angelman syndrome. The recent discovery of Ube3a substrates such as Arc (activity-regulated cytoskeletal protein) provides new insight into the mechanisms underlying the synaptic function and plasticity deficits caused by the loss of Ube3a. In addition to identifying Ube3a substrates, there have also been recent advances in understanding UBE3A's integrated role in the neuronal repertoire of genes and protein interactions. A developmental picture is now emerging whereby UBE3A gene dosage on chromosome 15 alters synaptic function, with deficiencies leading to Angelman syndrome and overexpression associated with classic autism symptomatology

A GAP in Form and Function: The Rac-GAP Alpha2-Chimaerin Regulates Dendrite Morphogenesis and Synaptic Plasticity.

A remarkable feature of the central nervous system is the highly choreographed wiring of neuronal circuits, which provides the structural and functional framework for cognitive abilities in humans. One critical parameter in this process is the elaboration of axonal and dendritic arbors as their size dictates the number of synaptic inputs a neuron can form. Such exquisite connectivity relies on the morphological complexity of the individual neuron. In neurons, cytoskeletal regulators dictate the extent of axonal and dendritic arborization. At the synapse, molecular mechanisms alter cytoskeletal dynamics in response to extracellular cues, thus linking neuronal morphological plasticity to synaptic activity. A central goal in Neuroscience is to elucidate the molecular mechanisms that regulate intracellular cytoskeletal rearrangement, which mediates morphological transformations in dendritic spines. Importantly, aberrant dendritic spine morphogenesis and synaptic function are unifying features in neurodevelopmental, neuropsychiatric and neurodegenerative diseases. Defects in EphR/ephrin signaling and Rac1 function at the synapse are implicated in the etiology of neurological disorders that exhibit dendritic spine defects. My dissertation contains three independent studies that focus on the molecular players that link structural and functional mechanisms at the synapse. First, I identified that the Rac-GAP, α2-chimaerin, is a key regulator of convergent signaling pathways required for normal dendritic spine morphogenesis and synaptic function. Second, I demonstrate that the loss of α2-chimaerin alters proliferation in the adult dentate gyrus that may correlate to a reduction in basal anxiety in rodents. Third, I characterized an excitatory ionotropic GABA receptor in the nematode Caenorhabditis elegans, and determined it plays a role in extrasynaptic spillover transmission. Together, these data shed new insight into the molecular mechanisms that regulate dendritic spine morphogenesis and synaptic function, and demonstrate that α2-chimaerin is poised at the nexus of critical signaling pathways to transduce extracellular synaptic signaling to intracellular regulation of dendritic spines and synapses.PhDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/120893/1/cmvaldez_1.pd