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Molecular mechanisms of synapto-dendritic cargo trafficking

Final Report Summary - NEUROTRAF (Molecular mechanisms of synapto-dendritic cargo trafficking)

Activity-dependent modulation of synapses is the core mechanism of synaptic plasticity and underlies learning and memory processes in the brain. Understanding dynamic changes at synapses requires a deeper insight in the molecular machinery involved in the transport and trafficking of postsynaptic proteins. Recent studies suggest that the cytoskeleton (microtubule and actin) and molecular motors (kinesin, dynein and myosin) play an essential role in the targeting and delivery of cargos to synapses. Moreover, it has been shown that defects in synaptic cargo transport are common feature of many human neurodegenerative and psychiatric diseases. One of the limiting factors to study synaptic cargo trafficking in living neurons is the lack of appropriate molecular and imaging tools. Recent developments in super-resolution microscopy as well as the progress made in nanotechnology yield many new possibilities to study synaptic trafficking processes in neurons. In this proposal, we aimed to address key issues in synapto-dendritic protein transport by developing novel molecular tools and imaging systems in living neurons. These tools will be utilized to answer a number of fundamental questions: 1) What is the contribution of specific motor proteins to dendritic transport? 2) Do microtubule motors enter dendritic spines and transport cargos in and out? 3) What is the mechanism of cargo movement between neighboring spines? The expected outcomes of this project will contribute fundamental insights into trafficking processes in neurons and deepen our understanding of the molecular dynamics of synapses.

Since the beginning of the project our work has been focused on producing tools and establishment of a new methodology. Namely, we cloned and tested different expression constructs for motor proteins and for targeting sequences (mitochondria, peroxisomes, endosomes) fused to chemical- or light-inducible heterodimetization systems; optimize the functionalization, intracellular delivery and controlled targeting of Qdots and perform motor-cargo coupling assays in fibroblast cells and primary hippocampal neurons. During the same period I contributed to implementing super-resolution microscopy (dSTORM) in our Department, which helped us to discover a novel aspects of microtubule organization in dendrites. During the second half of the funding period we begin with preparation of technical manuscripts describing developed methodology. At the same time we used some of these techniques in combination with high-end fluorescence imaging and biochemical methods to study two biological questions dealing with i) role calcium signalling in activity-dependent dynamics of dendritic spines and ii) with a new mechanism of controlling the activity of subset kinesin motors via the Goldberg-Shprintzen syndrome (GOSH) protein KBP. The main outcomes of these projects are described below:

1. The nanobody-QDots approach opens up new possibilities for the use of nano- or intrabodies directed against endogenous neuronal proteins to link QDots and the target molecule. This will allow to study and steer intracellular processes by either labeling or clustering and creating a local hub of specific molecules or to analyze behavior of motor-coupled cargo in neurons. Manuscript is in preparation.

2. We have developed nanobodies against tubulin to achieve super-resolution imaging of microtubules with a decreased effective diameter. Using these probes we can resolve dendritic microtubules in hippocampal neurons allowing novel insights into mechanisms underlying dendrite formation and maintenance. Manuscript is submitted.

3. Filamentous actin, representing the major cytoskeletal components of dendritic spines, is involved in structural plasticity of spines as well as protein and vesicle transport. With the use of biochemical and biophysical and fluorescent microscopy approaches we deciphered a novel molecular mechanism by which a neuronal Ca2+-sensor caldendrin can impact on actin dynamics in spines. The work is far advanced and will be completed soon.

4. Defects in cargo transport can lead to various neurological diseases. One of such examples is homozygous nonsense mutations in the gene encoding KBP which was linked to Goldberg-Shprintzen (GOSH) syndrome, a severe neurological disorder characterized by mental retardation, microcephaly and Hirchsprung disease. In collaborative work from our Department we show that the protein KBP regulates motor activity by preventing kinesin movement along microtubules. Our study uncovers a novel mechanism for regulating the activity of kinesin motors and suggests that altered synaptic vesicle trafficking contributes to GOSH pathogenesis. This work has been submitted.

5. In addition to the main project, I contributed to several papers published by my previous and current labs. This work is related to the proposed questions and support by FP7 and Marie-Curie IEF is acknowledged.

As a final result of this project we developed methodology for controlled intracellular transport assays and super-resolution microscopy, which will be made publically available and find a broad application by scientific community. We have used these methods to address fundamental questions connecting our understanding of molecular mechanisms of neuronal protein transport with the pathology of severe neurological disorder (GOSH syndrome).