Induction and Maintenance of Synaptic Plasticity

Synaptic long-term modifications following neuronal activation are believed to be at the origin of learning and long-term memory. Recent experiments suggest that these long-term synaptic changes are all-or-none switch-like events between discrete states of a single synapse. The biochemical network involving calcium/calmodulin-dependent protein kinase II (CaMKII) and its regulating protein signaling cascade has been hypothesized to durably maintain the synaptic state in form of a bistable switch. Furthermore, it has been shown experimentally that CaMKII and associated proteins such as protein kinase A and calcineurin are necessary for the induction of long-lasting increases (long-term potentiation, LTP) and/or long-lasting decreases (long-term depression, LTD) of synaptic efficacy. However, the biochemical mechanisms by which experimental LTP/LTD protocols lead to corresponding transitions between the two states in realistic models of such networks are still unknown. We present a detailed biochemical model of the calcium/calmodulin-dependent autophosphorylation of CaMKII and the protein signaling cascade governing the dephosphorylation of CaMKII. As previously shown, two stable states of the CaMKII phosphorylation level exist at resting intracellular calcium concentrations. Repetitive high calcium levels switch the system from a weakly- to a highly phosphorylated state (LTP). We show that the reverse transition (LTD) can be mediated by elevated phosphatase activity at intermediate calcium levels. It is shown that the CaMKII kinase-phosphatase system can qualitatively reproduce plasticity results in response to spike-timing dependent plasticity (STDP) and presynaptic stimulation protocols. A reduced model based on the CaMKII system is used to elucidate which parameters control the synaptic plasticity outcomes in response to STDP protocols, and in particular how the plasticity results depend on the differential activation of phosphatase and kinase pathways and the level of noise in the calcium transients. Our results show that the protein network including CaMKII can account for (i) induction - through LTP/LTD-like transitions - and (ii) storage - due to its bistability - of synaptic changes. The model allows to link biochemical properties of the synapse with phenomenological 'learning rules' used by theoreticians in neural network studies

Translational switch for long-term maintenance of synaptic plasticity

Memory can last a lifetime, yet synaptic contacts that contribute to the storage of memory are composed of proteins that have much shorter lifetimes. A physiological model of memory formation, long-term potentiation (LTP), has a late protein-synthesis-dependent phase (L-LTP) that can last for many hours in slices or even for days in vivo. Could the activity-dependent synthesis of new proteins account for the persistence of L-LTP and memory? Here, we examine the proposal that a self-sustaining regulation of translation can form a bistable switch that can persistently regulate the on-site synthesis of plasticity-related proteins. We show that an αCaMKII–CPEB1 molecular pair can operate as a bistable switch. Our results imply that L-LTP should produce an increase in the total amount of αCaMKII at potentiated synapses. This study also proposes an explanation for why the application of protein synthesis and αCaMKII inhibitors at the induction and maintenance phases of L-LTP result in very different outcomes

Role of action dynamics in the cooperative maintenance of synaptic plasticity

Tese de mestrado em Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2020As formas de plasticidade sináptica dependentes de atividade, tal como a potenciação de longa duração (LTP, do inglês long-term potentiation) e a depressão de longa duração (LTD, do inglês longterm depression) são mecanismos celulares associados aos processos de memória e aprendizagem. A LTP é a forma de plasticidade sináptica dependente de atividade mais estudada. O mecanismo molecular envolvido na indução de LTP é complexo, e envolve a ativação de diversas vias de sinalização. A indução é iniciada com a entrada de iões de cálcio para os neurónios pós-sinápticos através dos recetores de glutamato N-Metil-D-Aspartato (do inglês NMDA, N-methyl-D-aspartate). A entrada de cálcio através deste recetor conduz a ativação da proteína quinase II dependente de cálcio e calmodulina (do inglês CaMKII, Calcium /Calmodulin-dependent protein kinase II). Uma vez ativada, esta proteína conduz a translocação dos recetores alfa-amino-3-hidroxi-metil-5-4-isoxazolpropiónico (do inglês AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) para a sinapse, potencializando a transmissão sináptica. Este mecanismo está geralmente associado à indução de uma forma de LTP transiente. Para que as alterações na eficácia sejam mantidas, estas necessitam de mecanismos de transcrição e tradução de modo a ocorrer a síntese de proteínas associadas a fenómenos de plasticidade (PRPs-do inglês plasticty-related proteins) e de um marcador sináptico que irá capturar estas proteínas. Esta hipótese foi sugerida pela primeira fez em 1997 pelos investigadores Uwe Frey e Richard Morris, sendo classificada de hipótese da marcação e captura sináptica (do inglês STC, Synaptic Tagging and Capture hypothesis). Dado que as PRPs são apenas sintetizadas em resultado de uma tetanização forte, enquanto que a exibição do marcador pelas sinapses ocorre em ambas as tetanizações (fraca ou forte), fenómenos de cooperação e competição sináptica podem ocorrer no contexto da hipótese de STC. A cooperação sináptica define-se pela estabilização das formas transientes de LTP através da partilha de PRPs entre sinapses. As sinapses estimuladas com uma tetanização fraca podem cooperar com as sinapses estimuladas com uma tetanização forte permitindo deste modo a sua estabilização através da partilha de PRPs disponíveis. Por outro lado, a competição sináptica ocorre quando existe uma menor disponibilidade de proteínas ou maior número de sinapses ativadas, e por consequência mais marcadores disponíveis para a capture de PRPs. Apesar de a hipótese do STC ter sido descrita há mais de 20 anos, a identidade molecular do marcador sináptico continua a ser alvo de intensa investigação. O citoesqueleto de actina tem sido colocado com um potencial candidato para o papel de marcador sináptico. O citoesqueleto de actina apresenta um papel importante quer na plasticidade funcional como na plasticidade estrutural. Estudos anteriores sugerem que a modulação da rede de actina, através de fármacos que afetam a sua polimerização ou despolimerização, interfere com as formas persistentes da LTP. Por outro lado, estudos revelam que a modulação da dinâmica da actina conduz a alterações estruturais nas espinhas dendríticas. A dinâmica da actina envolve uma via de sinalização complexa em várias proteínas que se podem ligar a ela, chamadas de proteínas que se ligam à actina (do inglês ABP, Actin-binding proteins). Uma destas proteínas é CaMKII cuja sua ativação conduz à modulação de diversas moléculas a jusante, tais como Cdc42, um membro da família das GTPases. Esta molécula tem como principais caraterísticas a sua capacidade de modelar o citoesqueleto actina de uma forma dependente da atividade e o facto de esta ser espacialmente limitada às espinhas dendríticas estimuladas. Dadas as características da Cdc42, nós propomos que esta molécula representa um papel importante na modulação do setting do marcador sináptico. Neste trabalho, pretende-se investigar o papel da Cdc42 na plasticidade sináptica, utilizando um inibidor seletivo da mesma (ML141). Nós estudámos a importância da ativação desta molécula na indução e expressão de formas transientes e persistentes de LTP, bem como o seu papel nos fenómenos de cooperação e competição sináptica. De modo a investigar as questões acima mencionadas, os potenciais excitatórios pós-sinápticos desencadeados pela estimulação dos colaterais de Schaffer foram registados no stratum radiatum da área CA1. Os nossos resultados demostram que a indução de formas transientes de LTP não são afetadas pela inibição da Cdc42. No entanto, a indução de formas persistentes de LTP requerem a ativação da Cdc42. Por outro lado, nós demonstramos que a ativação da Cdc42 é também necessária para a manutenção da plasticidade, no entanto a sua ativação apenas é necessária dentro de uma janela temporal especifica. Os nossos resultados demostraram que as formas persistentes de LTP podem ser destabilizadas se a inibição da Cdc42 ocorrer até 70 min após a indução de LTP. Nós também avaliamos o efeito da inibição da Cdc42 na cooperação e competição sináptica. Os nossos dados revelam que a inibição da Cdc42 conduz ao bloqueio da cooperação sináptica. As formas transientes de LTP não são capazes de cooperar com a formas persistentes de LTP, uma vez que a inibição da Cdc42 leva a destabilização do marcador sináptico, conduzindo deste modo a uma captura ineficiente de PRPs. No entanto, o bloqueio da cooperação sinápica pode ser revertido se a aplicação de ML141 for simultânea com a da citocalasina (um inibidor da polimerização da actina) ou se a aplicação de ML141 for simultânea com a suspensão da atividade sináptica. Na competição sináptica, os nossos resultados demostraram que a inibição de Cdc42 leva a estabilização de todos os inputs ativados. Em suma, os nossos resultados fornecem fortes evidências de que a Cdc42 desempenha um papel importante na hipótese do STC. Os resultados obtidos demostraram que a inibição do Cdc42 compromete a indução e a manutenção da LTP, assim como interfere com os mecanismos de cooperação e competição, através da modulação do citoesqueleto de actina.Activity-dependent changes in synaptic efficacy such as long-term potentiation (LTP) are widely accepted to be cellular mechanisms underlying memory and learning processes. Upon induction, LTP can be expressed as a transient or as a maintained form, depending on the stimulation strength. According to the Synaptic Tagging and Capture hypothesis (STC), the maintenance of activitydependent changes are depended on the interplay between an input-specific synaptic tag and the capture of plasticity-related proteins (PRPs). Given that maintained forms of LTP depend on PRPs capture, the activated synapses can undergo either synaptic cooperation or competition, depending on proteins availability. Actin cytoskeleton dynamics has been suggested as a major candidate for the synaptic tag role. Actin plays a critical role in synaptic plasticity, linking functional and structural plasticity. Induction of LTP leads to activation of CaMKII, which in turn leads to activation of several small GTPases such as Cdc42, a molecule involved in actin cytoskeleton remodeling during plasticity induction. Cdc42 modulates actin network in an activity-dependent manner and once activated Cdc42 is spatially restricted to the stimulated spine. Actin network assembling and disassembling leads to structural changes on dendritic spines and pharmacological modulation of the actin dynamics affects LTP maintenance, suggesting that the remodeling of the actin provides the molecular signal (synaptic tag) for PRPs capture. Here, we aim to assess the molecular mechanisms underlying the setting for the synaptic tag. To do this, we use a specific inhibitor of Cdc42 activation (ML141) and we investigate the role of Cdc42 activation in the synaptic tag modulation during LTP induction and expression, and also during synaptic cooperation and competition. Our results showed that inhibition of Cdc42 for 30 min does not interfere with the induction of transient forms of LTP, while the induction of persistent forms of LTP are Cdc42 activation-dependent. Moreover, our results showed that maintained forms of LTP can be destabilized if Cdc42 inhibition occurs within a specific time-window. Inhibition of Cdc42 70 min upon LTP induction does not interfere with LTP maintenance. We also test the role of Cdc42 activation in both synaptic cooperation and competition. We showed that inhibition of Cdc42 blocks the ability of synapses to cooperate, however the synaptic capture impairment induced by ML141 application was reverted when an actin polymerization inhibitor (cytochalasin) was added or when activity was suspended. In synaptic competition, we showed that inhibition of Cdc42 leads to the maintenance of all stimulated inputs. Our data showed that activation of Cdc42 is a key regulator of the synaptic tag modulation. We showed that inhibition of the Cdc42 clearly compromises LTP induction and maintenance, and both synaptic cooperation and competition were also affected. Together, our data provide strong evidence that supports the actin cytoskeleton as a potential synaptic tag

Tubulin tyrosination regulates synaptic function and is disrupted in Alzheimer's disease

: Microtubules play fundamental roles in the maintenance of neuronal processes and in synaptic function and plasticity. While dynamic microtubules are mainly composed of tyrosinated tubulin, long-lived microtubules contain detyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination cycle is a key player in the maintenance of microtubule dynamics and neuronal homeostasis, conditions which go awry in neurodegenerative diseases. In the tyrosination/detyrosination cycle, the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidases and re-added by tubulin tyrosine ligase. Here we show that tubulin tyrosine ligase hemizygous mice exhibit decreased tyrosinated microtubules, reduced dendritic spine density, and both synaptic plasticity and memory deficits. We further report decreased tubulin tyrosine ligase expression in sporadic and familial Alzheimer's disease, and reduced microtubule dynamics in human neurons harboring the familial APP-V717I mutation. Finally, we show that synapses visited by dynamic microtubules are more resistant to oligomeric amyloid β peptide toxicity and that expression of tubulin tyrosine ligase, by restoring microtubule entry into spines, suppresses the loss of synapses induced by amyloid β peptide. Together, our results demonstrate that a balanced tyrosination/detyrosination tubulin cycle is necessary for the maintenance of synaptic plasticity, is protective against amyloid β peptide-induced synaptic damage, and that this balance is lost in Alzheimer's disease, providing evidence that defective tubulin retyrosination may contribute to circuit dysfunction during neurodegeneration in Alzheimer's disease

Visualization of the distribution of autophosphorylated calcium/calmodulin-dependent protein kinase II after tetanic stimulation in the CA1 area of the hippocampus

Autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) at threonine-286 produces Ca2+-independent kinase activity and has been proposed to be involved in induction of long-term potentiation by tetanic stimulation in the hippocampus. We have used an immunocytochemical method to visualize and quantify the pattern of autophosphorylation of CaMKII in hippocampal slices after tetanization of the Schaffer collateral pathway. Thirty minutes after tetanic stimulation, autophosphorylated CaM kinase II (P-CaMKII) is significantly increased in area CA1 both in apical dendrites and in pyramidal cell somas. In apical dendrites, this increase is accompanied by an equally significant increase in staining for nonphosphorylated CaM kinase II. Thus, the increase in P-CaMKII appears to be secondary to an increase in the total amount of CaMKII. In neuronal somas, however, the increase in P-CaMKII is not accompanied by an increase in the total amount of CaMKII. We suggest that tetanic stimulation of the Schaffer collateral pathway may induce new synthesis of CaMKII molecules in the apical dendrites, which contain mRNA encoding its alpha-subunit. In neuronal somas, however, tetanic stimulation appears to result in long-lasting increases in P-CaMKII independent of an increase in the total amount of CaMKII. Our findings are consistent with a role for autophosphorylation of CaMKII in the induction and/or maintenance of long-term potentiation, but they indicate that the effects of tetanus on the kinase and its activity are not confined to synapses and may involve induction of new synthesis of kinase in dendrites as well as increases in the level of autophosphorylated kinase

State based model of long-term potentiation and synaptic tagging and capture

Recent data indicate that plasticity protocols have not only synapse-specific but also more widespread effects. In particular, in synaptic tagging and capture (STC), tagged synapses can capture plasticity-related proteins, synthesized in response to strong stimulation of other synapses. This leads to long-lasting modification of only weakly stimulated synapses. Here we present a biophysical model of synaptic plasticity in the hippocampus that incorporates several key results from experiments on STC. The model specifies a set of physical states in which a synapse can exist, together with transition rates that are affected by high- and low-frequency stimulation protocols. In contrast to most standard plasticity models, the model exhibits both early- and late-phase LTP/D, de-potentiation, and STC. As such, it provides a useful starting point for further theoretical work on the role of STC in learning and memory