Blanc SVSE 2 - Sciences de la vie, de la santé et des écosystèmes : Biologie cellulaire, développement

Cellular functions of STOP proteins: towards a functional and molecular understanding of microtubule effects on synaptic transmission and neuronal organisation – CBioS

STOP/MAP6 proteins and microtubules: crucial for neurons?

Towards an understanding of the molecular and functional role of microtubules and proteins STOP/MAP6 in synaptic transmission and neuronal organization

Define cellular and biochemical functions of STOP/MAP6 proteins

MAP6 proteins are major effectors of microtubule whose deletion in mice leads to serious impairment of higher mental functions with cognitive defects sensitive to antipsychotics. STOP KO mice is widely used to study different aspects of the pathophysiology of schizophrenia.<br />The challenge now is to study the cellular and molecular properties of proteins MAP6 to establish the links that connect these properties and higher mental functions.<br />We analyze:<br />-Dynamics and plasticity of growth cones and dendritic spines<br />-Dependent signaling cascades of MAP6 during axonal guidance<br />-Axonal transport<br />-Recruiting protein during activation of the synapse<br />-Basic properties of proteins MAP6 in the interaction with microtubules and actin

We grow neurons from embryos deficient for MAP6/STOP and compare their properties to wild type neurons. By fluorescence imaging we analyze various parameters : microtubule dynamics, axonal transport, differentiation.
We produce recombinant proteins in large quantities and analyze their properties in vitro vis-à-vis of microtubules or actin

-The study of in vitro differentiation of neurons STOP ko was conducted, it was possible to demonstrate
* An early differentiation
* An abnormal number of dendritic spines
* A lack of sensitivity to the positive action of semaphorin 3E
-The study of calcium channels showed that the amount of functional N-type channels and P / Q in the membrane is reduced
The 3-STOP/MAP6 isoforms were produced. The study of MAP6-F on the cold stability of microtubules has been finalized.
Thus it was shown that the protein MAP6-F is a «sensor« of cold and it changes its conformation depending on temperature to bind and protect microtubules from depolymerization

Continue the molecular and cellular characterization of MAP6/STOP proteins. Define functional motifs.

Delphin C, Bouvier D, M Seggio, Couriol E, Saoudi Y, Denarier E, Bosc C, Valiron O, Arnal I, and Annie Andrieux. Fibroblastic Microtubule Associated Protein 6 (MAP6-F) is a temperature-sensor That Directly Protects and binds to microtubules from cold-induced depolymerization. J Biol Chem 2012

Microtubules are prominent components of neurons; historically, they have been mainly regarded as house keeping structures and little attention has been devoted to their possible involvement in synaptic transmission or in integrated brain functions. Our group has provided one of the first experimental evidence for a role of microtubules in mental functions by developing a mouse model deficient for microtubule associated proteins known as STOPs. STOP-deficient mice show severe behavioural deficits that react positively to antipsychotic drugs such as neuroleptics. In our initial studies, these behavioural deficits could be associated with synaptic defects affecting both short-term and long-term synaptic plasticity. Recently we have found that STOP-null mice also show global reduction of the brain mass, associated with widespread deficits in axonal extensions, whereas the number of neurons seemed unaffected. Axonal anomalies are particularly conspicuous in the fornix tract whose posterior part is lacking in STOP-deficient mice.
The present application is based upon ongoing work in the laboratory that indicates that STOP-null mice phenotypes correlate with severe cellular anomalies affecting axonal morphogenesis and guidance, positioning of various membrane receptors and composition of post-synaptic densities. Moreover, the intrinsic behaviour of the growth cone is probably affected in cultured STOP-null neurons, whose axons are often tortuous and ramified. These anomalies may reflect a role of STOP in regulating growth cone cytoskeletal assemblies.
The posterior fornix is composed of axons of the neurons located in the subiculum. Interestingly, our current data indicate a complete lack of reactivity of STOP-deficient neurons from the subiculum to Semaphorin 3E, a major axonal guidance factor involved in fornix extension. We are currently testing neurons from other areas of the brain to ascertain general STOP requirement in semaphorin signalling.
At the subcellular level, we noticed that axonal vesicle transport in STOP-null neurons was affected, more precisely the dynein-dependent retrotransport. It is possible that anterotransport is affected as well since we measured deficits in functional calcium channels (responsible for neurotransmitter release) and TrkB receptors (involved in BDNF signalling) in the plasma membrane. With the help of electronic microscopy, we also observed a marked reduction in the thickness of the post synaptic densities (PSDs) in CA3 hippocampal synapses, involving an apparent defect of the synaptic activation-dependent protein import in STOP-null synapses. STOPs themselves are demonstrated core components of PSDs. We are currently testing if other proteins are lacking in the PSDs of activated STOP-null synapses, with special focus on CaMKinase II, which is a major protein imported in PSDs upon synaptic activation and linked to STOPs in several ways.
Finally, we have identified direct - or indirect - STOP partners, in addition to microtubules. The most relevant to the major cellular phenotypes identified include actin, CaMKII, CRMPs (Collapsin Response Mediator proteins) which are involved in semaphorin signalling, Tctex-1 which is a subunit of the retrograde motor dynein also involved in calcium signalling, and PSD 95, a key protein of synaptic plasticity. The exact role of these partners will be investigated within each relevant phenotype described above.

In the proposed program, we intend to use the recently identified cellular phenotypes of STOP-null neurons and new knowledge gathered about STOP partners to assess the role of STOPs in the major neuronal functions affected in STOP-null neurons and identify corresponding anomalies in defined molecular systems. Such data should help unravel how microtubules affect integrated brain functions in mammals.

Project coordination


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 400,000 euros
Beginning and duration of the scientific project: - 36 Months

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