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Final Report Summary - DYNAMICMTINSPINES (The role of dynamic microtubule in the structure and function of dendritic spines)

Neurons are among the most complex and polarized cells in the human body. They make use of efficient transport processes to cover the immense distances between cell body and outer neuronal regions. An elaborated interplay between the cytoskeleton (microtubules and actin) and molecular motors (kinesin, dynein and myosin) assures the delivery of cargo and organells to specific cellular compartments. The tight control of neuronal transport has been shown to be essential in the developing organism, as well as in adult cells in neuronal functions such as synaptic plasticity. The importance of this system is illustrated by the growing number of human neurodegenerative and psychiatric diseases that could be linked to defects in neuronal transport processes.

Microtubules are long tubular structures and can be regarded as the “high ways” of neuronal long distance transport. The intrinsic asymmetry of the microtubule lattice is recognized by microtubule-based motor proteins and defines their direction of transport. While neurons generally exhibit a high microtubule density, the polarity of microtubules significantly differs between axons and dendrites. In dendrites the polarity of microtubules is mixed and a distinct proportion of microtubule remains in a dynamic state. Previous studies from our lab have demonstrated that dynamic microtubules transiently enter dendritic spines, which represent the postsynaptic part of the excitatory synapse. The main objective of this project has been to identify the neuronal signals and mechanisms that facilitate microtubule entries in spines and reveal their role for synaptic function.

Up to now, studies on neuronal microtubules have almost exclusively been performed on dissociated neuron cultures. While this model system is advantageous for experimental manipulations and high resolution imaging, it also possesses a risk of culturing artefacts as a consequence of the in vitro conditions. Therefore, it was of great importance to initially confirm previous findings on microtubule dynamics in dendrites by using a different model system. We made use of cultured hippocampal slices transduced with lentivirus, which more closely resembles in vivo conditions, and recorded microtubule dynamics in this organotypic cellular environment. Our data confirmed the polarity differences of microtubules between axons and dendrites and additionally provided evidence for a new model on the establishment of cellular polarity during neuronal development. In collaboration with the group of Anthony Holtmaat (Geneva) we extended this study to 2-photon recordings in anesthetised living mice and were able to analyse for the first time microtubule dynamics in the intact central nervous system. The study has been recently published (Yau and Schätzle et al., 2015, JNS) and the reviewers have highlighted that it “makes an important contribution to our basic understanding of how the neuronal cytoskeleton is organized”.

Interestingly, microscopic observations in both organotypic slice cultures and in living mice showed clear evidence for the targeting of dynamic microtubules to dendritic spines. In the following we applied a detailed study on microtubule invasions in spines making use of cultured slices and dissociated neurons depending on the experimental requirements. The main outcomes are briefly described below:

1) We characterized the subset of dynamic microtubules that enters dendritic spines for multiple parameters. Even though slight variations between cultured neurons and slices were observed we were able to conclude that microtubule targeting of spines are common phenomena in the dendrites of mammalian neurons. Next, we focused on the regulation of microtubule invasions in order to exclude that these events occur randomly.

2) Pharmacological experiments revealed specific synaptic signals that can trigger microtubule invasions in spines. Our findings suggested that spine targeting is regulated on the level of individual synapses and we performed a set of experiments to verify this hypothesis. In addition, we worked on identifying the underlying signalling cascade translating synaptic signals in cellular responses that facilitate microtubule entries in spines.

3) Using a combination of high-resolution live imaging and pharmacological treatments, we identified actin as the key downstream target and effector of the spine entry targeting mechanism. In the meantime, this finding has been confirmed by an independent study using a similar approach. A screen for actin and microtubule interacting proteins provided evidence for a specific cellular mechanism that promotes microtubule entries in spines. A manuscript summarizing our findings on the triggering signals and the mechanism of microtubule entries in spines is in preparation.

4) Finally, we shifted our attention towards the functional implications of microtubule invasions in spines for synaptic function. The general importance of microtubules for dendritic transport processes also suggested a role in synaptic cargo transport. Pharmacological induced blockade of microtubule dynamics decreased the entry and exit speeds of endosomes in spines indicating that this cargo can be transported by microtubules. Next, we applied an inducible MT-dependent trafficking assay and observed increased targeting of Rab11 positive endosomes from the dendritic shaft to spines. In contrast, experimentally induced removal of recycling endosomes from spines affected spine morphology and function, demonstrating the importance of precise trafficking for dendritic spines. The results of this study were recently published together with additional findings on cargo trafficking in dendritic spines (Esteves da Silva et al., 2015, Cell Reports).

This project was aimed to better understand the phenomena of microtubule invasions in dendritic spines. The most important findings of this study demonstrated that this process is regulated by synaptic activity and can affect the composition of postsynapses via microtubule-based transport. We anticipate that our results will be of interest to scientists from academia and industry working in the field of synaptic plasticity. The novel concept that dynamic microtubules in dendrites can directly contribute to plastic changes at synapses makes it an attractive research target for future studies in the context of learning and memory function of the brain. In the light of recently developed microtubule targeting drugs, initially developed for cancer therapy, this will be an interesting starting point for the development of new therapeutic approaches in the treatment of neurological diseases linked to intellectual deficits.

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