Ongoing Projects

Semaphorin Signalling

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Semaphorins are multifunctional proteins essential for embryonic development and for various physiological functions in the adult. They interact with members of the Plexin or Neuropilin families of transmembrane receptors, leading to the activation of a diverse range of intracellular signalling events (reviewed in Jackson and Eickholt, 2009; Eickholt, 2008). One of the hallmarks of Semaphorins is their ability to alter cytoskeletal dynamics, which is important for different cellular processes, including the guidance of neuronal growth cones and neural crest cells, for angiogenesis, but also for tumorigenesis and metastasis. One of the focuses of work by my group has been to understand the signalling events mediated by members of the Semaphorins, in the developing nervous system. We demonstrated, for example, that GSK3 activation is required for Sema3A-induced growth cone collapse in DRG neurons. This work also showed that GSK3 is maintained inactive growth cone and rapidly activated following Sema3A treatment. We further demonstrated that the tumour suppressor PTEN regulates GSK3 signalling in response to Sema3A. This work highlighted the importance of sub-cellular distributions of PTEN to control growth cone behaviour, an unexplored mechanism that is the foundation of a number of current projects (see below).

Regulation of the PTEN tumour suppressor in neurons

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumour suppressor that can inhibit proliferation and migration as well as control cell growth and apoptosis in a number of different cells. PTEN functions predominately through its lipid phosphatase activity, converting phosphatidylinositol 3,4,5-trisphosphate (PIP3) to phosphatidylinositol 4,5-bisphosphate, thereby directly antagonizing the activity of PI3K and its established downstream signalling pathways such as those involved in regulating the cytoskeleton and the translational machinery. PTEN is highly expressed in post-mitotic neurons and recent work indicates that de-regulation of PTEN affects important neuronal functions in the brain, which have been attributed to its role in controlling neuronal growth, synaptogenesis and synaptic plasticity. We set out identifying the determining cellular machinery controlling PTEN functions in neurons through proteomics approaches. Our work identified a requirement for functional actin-based motor proteins in the control of PI3K signalling, a mechanism involving a previously unknown association between PTEN and class V Myosins. We demonstrate that inactivation of MyosinV-transport function in CNS neurons induced - in line with known attributes of PTEN-loss - enlarged neuronal soma size and increased PI3K and mTor signalling. Our data characterise a novel myosin-based transport mechanism that regulates the function of the PTEN tumour suppressor and PI3K signalling (van Diepen et al., 2009).

Functional analyses of the actin regulator Drebrin in the nervous system

Drebrin mouseThe actin-binding protein Drebrin is involved in the regulation of actin filament organization, especially during the formation of neurites and cell protrusion of motile cells. Drebrin is also enriched in dendritic spines, where it controls spine morphology and is part of the large postsynaptic protein complex regulating synaptic transmission. Progressive loss of Drebrin has been linked to Mild Cognitive Impairment, a transitional state between healthy aging and mild dementia. Major loss of Drebrin in dendritic spines is characteristically found in patients with Alzheimer's disease and Down's syndrome. Thus, Drebrin is a salient candidate protein involved in transducing transient synaptic dysfunction into irreversible spine degeneration and progressive cognitive deficits. However, the specific mode according to which DBN is regulated remains largely unknown.

We have recently characterized the interaction of the PTEN tumor suppressor with DBN: PTEN binds DBN and negatively regulates DBN phosphorylation at S647 (Kreis et al., 2013). Our results identify a novel mechanism by which PTEN is required to maintain DBN phosphorylation at dynamic range. To study the precise role of Drebrin and the significance of Drebrin phosphorylation in vivo, we have recently established conditional knockout mouse lines and ongoing characterisation confirms Drebrin loss (Drebrin A and Drebrin E) in dependence of Cre. We also developed a rapid and reversible system to express exogenous Drebrin variants at physiological levels. These "knock-out / rescue" approaches will help us to gain comprehensive insight into the regulation of neuronal Drebrin, in particular, during synaptic plasticity and neurological disease.

PI3K and Synapses: PI3K Signalling and Spine Morphogenesis

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Synaptic transmission is closely linked to the morphology of dendritic spines, which are protrusions on the neuronal surface that receive most of the excitatory input. Rather than being stable structures, spines are dynamic and continuously undergo shape changes. Such changes in morphology are directly influenced by synaptic activity, intracellular ion concentrations and by external factors, and can have major impact on synapse function including synaptic efficacy. Spine shape is controlled by changes in the cytoskeleton which mainly consists of actin filaments. During spine morphogenesis, PI3K signalling has been shown to greatly affect normal spine shape. For example, activation of PI3K signalling causes increases in filopodia spine length, and, conversely, inhibition of this signalling pathway by overexpression of PTEN reduces both dendritic spine number and filopodia. The downstream signalling effectors of PI3K activity controlling dendritic spine morphogenesis are largely unknown. We identified an actin binding protein highly enriched in dendritic spines as novel PTEN interactor. We are currently testing if this protein-protein interaction may impinge on regulating actin dynamics and, together, may determine the shape and dynamic behaviour of dendritic protrusions.

Akt isoform-specific phosphorylation in nervous system development and disease

kt analysesAkt is a threonine/serine kinase downstream of PI3K signaling, and critical for regulation of cell growth, survival, proliferation and differentiation. Deregulation of Akt activity due to aberrant PI3K signaling has been linked to the progression of various pathological conditions including cancer and neurodevelopmental disorders. There are three isoforms encoded by three genes: Akt1, Akt2 and Akt3. An exact role of these isoforms in Akt signaling has not been completely established. Akt has three main phosphorylation sites, two activating ones at S473 and T308 (numbered according to Akt1) and a constitutive, stabilizing phosphorylation at T450. Western blot analyses of the different phosphorylation sites using phospho-specific antibodies is limited due to the lack of isoform specificity of antibodies. Most widely used techniques also suffer from limitations in their requirement for a large sample size. We use isoelectric focusing (IEF) to investigate isoform-specific phosphorylation states by differences in their isoelectric point (pI). Analysis of isoforms and individual phosphorylation states of native proteins by IEF occurs through separation in capillaries with rapid immobilization, allowing resolution and quantification using a single pan-specific antibody.

We established an assay for Akt to identify individual isoforms and their specific phosphorylation in growth factor (e.g. Insulin) stimulated or PI3K inhibitor treated cell lysates. This newly developed assay provides a basis to rapidly analyze cell lysates and tissues for their isoform-specific Akt phosphorylation and expression after stimuli or pathologic changes.

Britta Eickholt

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