Research

We use genetics, biochemistry, molecular, and cellular biology approaches to study neuronal development and functioning, with particular emphasis on the areas of microtubule cytoskeleton, protein trafficking and synaptic mechanisms. We tackle these questions using state-of-art techniques and in vitro, ex vivo, and in vivo systems.

Our research addresses four areas:

Synaptic protein transport

Synapses are composed of a presynaptic terminal, a synaptic cleft, and a postsynaptic specialization and are the structures through which neurons communicate. Recently enormous progress has been made in identifying the molecular components of synapses, particularly by using proteomic strategies and the principles governing their assembly. These studies have revealed that synapses are composed of hundreds of proteins, and that the specification of synaptic function, e.g. excitatory or inhibitory, at both pre- and post-synapses is achieved via the recruitment and assembly of particular protein complexes. Amongst the highly conserved molecules that are of critical importance for the initial formation and proper functioning of synapses are synaptic scaffolding proteins. Recently, synaptic scaffolding proteins have also been shown to associate with motor-protein complexes. These proteins function as adaptor molecules between molecular motors and synaptic receptors during the processes of cargo transport towards and/or from the synaptic specialization. Scaffolding proteins contain multiple domains for protein–protein interactions, which might enable motors to interact with large number of synaptic cargo, such as ion channels, adhesion molecules, receptors and other synaptic components. Studying the intracellular transport of synaptic cargo helps us to understand fundamental operational principles of synapse formation and functioning.

Recent evidence suggests that scaffolding proteins liprin-alpha and Glutamate Receptor Interacting Protein (GRIP) regulate synaptic cargo transport. Using cultured hippocampal neurons as a model system, we study the role of liprin-alpha and GRIP in synapse development, function and intracellular transport.

Motor-protein cargo adaptors

Microtubule-based motor proteins kinesin and dynein play a vital role in cellular trafficking and cell polarity by transporting cargoes to specific parts of the cell. How each motor protein selects its specific cargo and regulates binding and release is still and open question. Bicaudal-D (BICD) is well-characterized cargo adaptor for dynein-based transport, can regulate cargo selection and induces microtubule minus-end directed transport. Drosophila BicD is required for cytoplasmic dynein dependent mRNA transport during oocyte development and embryogenesis. We showed that the mammalian Bicaudal D homologues BICD1 and BICD2 bind with their N-terminal part to cytoplasmic dynein and dynactin and C-terminal part the small GTPase Rab6 which is involved in exocytotic vesicle trafficking. We propose a model in which BICD can recruit microtubule motors to Rab6-coated secretory vesicles. To view our BICD Bioclip click here

One major challenge is to understand how a cell coordinates all aspects of motor protein functions by coordinately regulating its cargo selection and binding/release of cargoes in a timely and spatially-coordinated manner. The lab is currently exploring the role of BICD in neuronal development and characterizing novel BICD-like proteins.

Neuronal cytoskeleton

Neurons display a characteristic microtubule organization distinct from dividing cultured cells. Most neuronal microtubules are not attached to the centrosome and form dense bundles running along the length of axons and dendrites. Individual microtubules do not extend along the entire length of neuronal processes; instead, microtubules fragments stabilized at their minus-ends form regularly spaced longitudinal arrays cross-linked by microtubule associated proteins (MAPs). Microtubule arrays within neuronal processes are highly organized with respect to their intrinsic polarity. Ultrastructural studies show that in axons, microtubules are generally long and uniformly oriented, with their plus-ends distal to the cell body, whereas in proximal dendrites microtubules are much shorter and exhibit mixed polarity. More distal thinner dendrites of higher order, however, contain unipolar microtubules oriented the same way as the axonal ones. This specialized microtubule organization has been recently captured in action by visualizing EB3-GFP in living neuronal cells. Since the major role of microtubules in mature neurons is to act as railways for the motor-based transport, distinct patterns of MT polarity orientation can generate asymmetries in the composition of each neuronal compartment by directing specific motor protein mobility. In this way, microtubules dynamics and polarity are most intimately related to neuronal morphology and functions.

The two microtubules ends are functionally distinct: the plus-ends (the fast growing ends in vitro) can rapidly switch between episodes of growth and shrinkage and the minus-ends are either stabilized or serve as MT depolymerization sites. A very specialized class of MAPs, which has recently received a lot of attention, is represented by MT plus-end tracking proteins (+TIPs). +TIPs comprise structurally unrelated proteins and are distinguished by their dynamic behavior: these proteins form transient comet-like accumulations at the growing microtubule plus-ends. Several +TIPs, including CLIPs, CLASPs, EBs, APC, p150Glued (the large subunit of dynactin complex) and Lissencephaly 1 (LIS1) have been shown to be important during several stages of neuronal development including migration, formation, growth and guidance of axons. Also proper communication between mature neuronal cells depends on +TIPs because of their involvement in positioning of organelles, receptors and channels. Not surprisingly +TIPs malfunction can lead to several nervous system-related disorders. We study the role of +TIPs in neuronal development and functioning.

Diseases linked to intracellular transport

Neuronal transport is the mechanism by which materials moved along the axon and dendrites. In an axon, this transport goes in two directions: in anterograde (or ‘forward’) transport, cargo is moved away from the cell body, and in retrograde (’backward’) transport, it is moved towards the cell body. For example, voltage-gated ion channels and receptors destined for the synapse are synthesized in the cell body and packaged into membrane-bound vesicles, which are then latched onto the transport machinery and moved along the axon to the nerve terminal. Neurotrophic factors, which are essential for maintenance of the neuron, bind to cell surface receptors and transported back towards the nucleus, where they alter gene expression. Hampering the transport of proteins within neurons may underlie several adult-onset neurodegenerative diseases, such as Huntington’s disease and the motor neuron degenerative disease Amyotrophic Lateral Sclerosis (ALS). Understanding how this intracellular transport is blocked in these diseases may offer targets for future therapy.

Recently, a missense mutation (P56S) in the gene encoding vesicle associated membrane protein (VAMP)-binding protein B (VAPB) has been identified in a familial form of ALS. VAP family proteins, VAPA and VAPB, are ubiquitously expressed type II integral membrane proteins which localize to the endoplasmic reticulum (ER) and pre-Golgi intermediates and have been proposed to regulate transport between the ER and the Golgi. Moreover, VAPs have been shown to target lipid-binding proteins carrying the FFAT motif to the ER. In yeast, the VAP-homologue Scs2 binds the FFAT motif and in absence of Scs2 the FFAT-containing proteins mislocalize to the cytoplasm. We have found that VAPB is abundant in motor neurons and that the P56S substitution causes aggregation of mutant VAPB in immobile tubular ER clusters, perturb FFAT-motif binding and traps endogenous VAP in mutant aggregates. We propose a model in which reduced levels of VAP family proteins result in decreased ER anchoring of lipid binding proteins and cause motor neuron degeneration.