The light chain of MAP1B: a novel modulator of AMPA receptor trafficking in hippocampal neurons

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-07-2014En el cerebro, la eficacia de la transmisión sináptica depende en gran medida del control dinámico ejercido sobre la adición a las sinapsis y la eliminación de las mismas de ciertas clases de receptores de neurotransmisores. Los receptores de glutamato de tipo AMPA (AMPARs) median, de forma mayoritaria, la transmisión sináptica excitatoria en el sistema nervioso central de los mamíferos. De hecho, la regulación de su tráfico intracelular constituye uno de los mecanismos principales por los cuales se modulan los fenómenos de plasticidad sináptica en las sinapsis hipocampales. En este trabajo nos hemos propuesto explorar la función de MAP1B, una proteína asociada a microtúbulos, en la regulación del tráfico de AMPARs en las neuronas piramidales CA1 de hipocampo. Mediante una combinación de herramientas moleculares, electrofisiología y microscopía confocal, hemos revelado una nueva función de la cadena ligera de MAP1B (MAP1B-LC) como elemento regulador del transporte intracelular de una población específica de AMPARs. Hemos podido determinar que la sobre-expresión de MAP1B-LC resulta en una reducción neta de la fracción móvil en dendritas de los AMPARs constituidos por la subunidad GluA2, y como consecuencia, en una acumulación disminuida en espinas, sin que los receptores formados por la subunidad GluA1 se vean afectados. En efecto, hemos podido comprobar que es la población endógena de receptores GluA2-GluA3 la que se ve afectada específicamente cuando se sobre-expresa MAP1B-LC, ya que su transporte constitutivo hacia las sinapsis, y por tanto, la transmisión sináptica basal, se ven reducidos en presencia de niveles incrementados de MAP1B-LC. Por otra parte, hemos demostrado que la distribución a lo largo de las dendritas de GRIP1, una proteína de ensamblaje que interacciona específicamente con las subunidades GluA2 y GluA3 y también con MAP1B-LC, se ve así mismo alterada como consecuencia de la sobre-expresión de MAP1B-LC. Así, por medio de mutantes de deleción de MAP1B-LC, hemos podido concluir que la unión de MAP1B-LC a GRIP1 junto con su interacción con los microtúbulos es esencial para regular la expresión en superficie y la presencia en las sinapsis de la población GluA2-GluA3 de AMPARs, y por consiguiente, su contribución a la transmisión sináptica basal en neuronas hipocampales CA1. Es importante destacar que el modelo que proponemos asigna, por primera vez, un significado funcional a la interacción entre MAP1B-LC y GRIP1.The strength of synaptic transmission in the brain relays largely on the controlled addition and removal of neurotransmitter receptors to and from synapses. AMPA-type glutamate receptors (AMPARs) mediate the vast majority of excitatory transmission in the mammalian central nervous system. Their regulated trafficking has been proposed to be one of the major mechanisms underlying the expression of synaptic plasticity at hippocampal synapses. In this work, we have explored the potential role of a microtubule-associated protein, MAP1B, in the fine-tuning of AMPAR trafficking in CA1 hippocampal neurons. Using a combination of molecular tools, electrophysiology and confocal microscopy, we reveal a novel role of the light chain of MAP1B (MAP1B-LC) as a key player in the subcellular sorting of a specific population of AMPARs. We demonstrate that MAP1B-LC over-expression results in a net reduction of the mobile population in dendrites and their accumulation in spines of recombinant GluA2 AMPARs, whereas the dendritic trafficking and delivery to spines of recombinant GluA1 AMPARs is unaltered. Indeed, we show that MAP1B-LC targets specifically the endogenous GluA2-GluA3 population of AMPARs, as their constitutive cycling toward synapses is impaired upon MAP1B-LC over-expression and consequently, basal synaptic transmission is decreased. We also demonstrate that the dendritic targeting of GRIP1, a specific interactor of GluA2/GluA3 subunits that also binds MAP1B-LC, is altered in the presence of enhanced levels of MAP1B-LC. Using deletion mutants of MAP1B-LC, we conclude that MAP1B-LC binding to GRIP1 together with its ability to interact with microtubules is essential to regulate the surface expression and presence at synapses of the GluA2-GluA3 population of AMPARs, and consequently, the degree to which they contribute to basal synaptic transmission in CA1 hippocampal neurons. Importantly, the model we propose assigns a functional meaning to the interaction between MAP1B-LC and GRIP1 for the first tim

Insulin regulates neurovascular coupling through astrocytes.

Circulating insulin enters the brain through mechanisms incompletely characterized. We now report that mice lacking insulin receptors (IR) in astrocytes (GFAP-IR KO mice) show blunted brain responses to insulin, uncoupling of brain blood flow with glucose uptake with concomitant changes in brain vasculature and glucose transporter 1 levels. IR-deficient astrocytes show increased expression of HIF-1α/VEGF, promote growth of co-cultured endothelial cells, display increased reactive oxidant species (ROS) and disturbed mitochondrial activity. Treatment with the antioxidant N-acetylcysteine (NAC), ameliorated high ROS levels, normalized angiogenic signaling, and mitochondrial function including mitochondrial glucose and oxygen sensors. In vivo treatment with NAC also normalized brain perfusion. Thus, insulin receptors in astrocytes regulate neuro-vascular coupling.pre-print4711 K

MAP1B Light Chain Modulates Synaptic Transmission via AMPA Receptor Intracellular Trapping.

The regulated transport of AMPA-type glutamate receptors (AMPARs) to the synaptic membrane is a key mechanism to determine the strength of excitatory synaptic transmission in the brain. In this work, we uncovered a new role for the microtubule-associated protein MAP1B in modulating access of AMPARs to the postsynaptic membrane. Using mice and rats of either sex, we show that MAP1B light chain (LC) accumulates in the somatodendritic compartment of hippocampal neurons, where it forms immobile complexes on microtubules that limit vesicular transport. These complexes restrict AMPAR dendritic mobility, leading to the intracellular trapping of receptors and impairing their access to the dendritic surface and spines. Accordingly, increasing MAP1B-LC expression depresses AMPAR-mediated synaptic transmission. This effect is specific for the GluA2 subunit of the AMPAR and requires glutamate receptor interacting protein 1 (GRIP1) interaction with MAP1B-LC. Therefore, MAP1B-LC represents an alternative link between GRIP1-AMPARs and microtubules that does not result in productive transport, but rather limits AMPAR availability for synaptic insertion, with a direct impact on synaptic transmission.pre-print7208 K

Neuronal p38α mediates synaptic and cognitive dysfunction in an Alzheimer’s mouse model by controlling β-amyloid production.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a severe and progressive neuronal loss leading to cognitive dysfunctions. Previous reports, based on the use of chemical inhibitors, have connected the stress kinase p38α to neuroinflammation, neuronal death and synaptic dysfunction. To explore the specific role of neuronal p38α signalling in the appearance of pathological symptoms, we have generated mice that combine expression of the 5XFAD transgenes to induce AD symptoms with the downregulation of p38α only in neurons (5XFAD/p38α∆-N). We found that the neuronal-specific deletion of p38α improves the memory loss and long-term potentiation impairment induced by 5XFAD transgenes. Furthermore, 5XFAD/p38α∆-N mice display reduced amyloid-β accumulation, improved neurogenesis, and important changes in brain cytokine expression compared with 5XFAD mice. Our results implicate neuronal p38α signalling in the synaptic plasticity dysfunction and memory impairment observed in 5XFAD mice, by regulating both amyloid-β deposition in the brain and the relay of this accumulation to mount an inflammatory response, which leads to the cognitive deficits.post-print1848 K

Astrocytic p38α MAPK drives NMDA receptor-dependent long-term depression and modulates long-term memory.

NMDA receptor-dependent long-term depression (LTD) in the hippocampus is a well-known form of synaptic plasticity that has been linked to different cognitive functions. The core mechanism for this form of plasticity is thought to be entirely neuronal. However, we now demonstrate that astrocytic activity drives LTD at CA3-CA1 synapses. We have found that LTD induction enhances astrocyte-to-neuron communication mediated by glutamate, and that Ca2+ signaling and SNARE-dependent vesicular release from the astrocyte are required for LTD expression. In addition, using optogenetic techniques, we show that low-frequency astrocytic activation, in the absence of presynaptic activity, is sufficient to induce postsynaptic AMPA receptor removal and LTD expression. Using cell-type-specific gene deletion, we show that astrocytic p38α MAPK is required for the increased astrocytic glutamate release and astrocyte-to-neuron communication during low-frequency stimulation. Accordingly, removal of astrocytic (but not neuronal) p38α abolishes LTD expression. Finally, this mechanism modulates long-term memory in vivo.post-print5316 K

Potentiation of amyloid beta phagocytosis and amelioration of synaptic dysfunction upon FAAH deletion in a mouse model of Alzheimer’s disease.

Background: The complex pathophysiology of Alzheimer’s disease (AD) hampers the development of effective treatments. Attempts to prevent neurodegeneration in AD have failed so far, highlighting the need for further clarification of the underlying cellular and molecular mechanisms. Neuroinflammation seems to play a crucial role in disease progression, although its specific contribution to AD pathogenesis remains elusive. We have previously shown that the modulation of the endocannabinoid system (ECS) renders beneficial effects in a context of amyloidosis, which triggers neuroinflammation. In the 5xFAD model, the genetic inactivation of the enzyme that degrades anandamide (AEA), the fatty acid amide hydrolase (FAAH), was associated with a significant amelioration of the memory deficit. Methods: In this work, we use electrophysiology, flow cytometry and molecular analysis to evaluate the cellular and molecular mechanisms underlying the improvement associated to the increased endocannabinoid tone in the 5xFAD mouse− model. Results: We demonstrate that the chronic enhancement of the endocannabinoid tone rescues hippocampal synaptic plasticity in the 5xFAD mouse model. At the CA3–CA1 synapse, both basal synaptic transmission and longterm potentiation (LTP) of synaptic transmission are normalized upon FAAH genetic inactivation, in a CB1 receptor (CB1R)- and TRPV1 receptor-independent manner. Dendritic spine density in CA1 pyramidal neurons, which is notably decreased in 6-month-old 5xFAD animals, is also restored. Importantly, we reveal that the expression of microglial factors linked to phagocytic activity, such as TREM2 and CTSD, and other factors related to amyloid beta clearance and involved in neuron–glia crosstalk, such as complement component C3 and complement receptor C3AR, are specifically upregulated in 5xFAD/FAAH−/− animals. Conclusion: In summary, our findings support the therapeutic potential of modulating, rather than suppressing, neuroinflammation in Alzheimer’s disease. In our model, the long-term enhancement of the endocannabinoid tone triggered augmented microglial activation and amyloid beta phagocytosis, and a consequent reversal in the neuronal phenotype associated to the diseasepost-print4206 K