Raphael Berges

Review of the Multiple Aspects of Neurofilament Functions, and their Possible Contribution to Neurodegeneration

Neurofilaments (NF) are the most abundant cytoskeletal component of large myelinated axons from adult central and peripheral nervous system. Here, we provide an overview of the complementary approaches, including biochemistry, cell biology and transgenic technology that were used to investigate the assembly, axonal transport and functions of NF in normal and pathological situations. Following their synthesis and assembly in the cell body, NFs are transported along the axon. This process is finely regulated via phosphorylation of the carboxy-terminal part of the two high-molecular-weight subunits of NF. The correct formation of an axonal network of NF is crucial for the establishment and maintenance of axonal calibre and consequently for the optimisation of conduction velocity. The frequent disorganisation of NF network observed in several neuropathologies support their contribution. However, despite the presence of NF mutations found in some patients, the exact relations between these mutations, the abnormal NF organisation and the pathological process remain a challenging field of investigation.

Neurofilaments Bind Tubulin and Modulate Its Polymerization

Neurofilaments assemble from three intermediate-filament proteins, contribute to the radial growth of axons, and are exceptionally stable. Microtubules are dynamic structures that assemble from tubulin dimers to support intracellular transport of molecules and organelles. We show here that neurofilaments, and other intermediate-filament proteins, contain motifs in their N-terminal domains that bind unassembled tubulin. Peptides containing such motifs inhibit the in vitro polymerization of microtubules and can be taken up by cultured cells in which they disrupt microtubules leading to altered cell shapes and an arrest of division. In transgenic mice in which neurofilaments are withheld from the axonal compartment, axonal tubulin accumulation is normal but microtubules assemble in excessive numbers. These observations suggest a model in which axonal neurofilaments modulate local microtubule assembly. This capacity also suggests novel mechanisms through which inherited or acquired disruptions in intermediate filaments might contribute to pathogenesis in multiple conditions.

A Tubulin Binding Peptide Targets Glioma Cells Disrupting Their Microtubules, Blocking Migration, and Inducing Apoptosis

Despite aggressive treatment regimes, glioma remains a largely fatal disease. Current treatment limitations are attributed to the precarious locations within the brain where such tumors grow, their highly infiltrative nature precluding complete resection and lack of specificity among agents capable of attenuating their growth. Here, we show that in vitro, glioma cells of diverse origins internalize a peptide encompassing a tubulin-binding site (TBS) on the neurofilament light protein. The internalized peptide disrupts the microtubule network, inhibits migration and proliferation, and leads to apoptosis. Using an intracerebral transplant model, we show that most, if not all, of these responses to peptide exposure also occur in vivo. Notably, a single intratumor injection significantly attenuates tumor growth, while neither peptide uptake nor downstream consequences are observed elsewhere in the host nervous system. Such preferential uptake suggests that the peptide may have potential as a primary or supplementary glioblastoma treatment modality by exploiting its autonomous microtubule-disrupting activity or engaging its capacity to selectively target glioma cells with other cell-disrupting cargos.

Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.

We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs), increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+) end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length and EB1 detyrosination/retyrosination cycle.

The effect of functionalizing lipid nanocapsules with NFL-TBS.40-63 peptide on their uptake by glioblastoma cells

We previously described a neurofilament derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters in glioblastoma cells where it disturbs the microtubule network both in vitro and in vivo. The aim of this study is to test whether this peptide can increase the targeted uptake by glioblastoma cells of lipid nanocapsules filled with Paclitaxel, and thus can increase their anti-proliferation in vitro and in vivo. Here, using the drop tensiometry we show that approximately 60 NFL-TBS.40-63 peptides can bind to one 50 nm lipid nanocapsule. When nanocapsules are filled with a far-red fluorochrome (DiD) and Paclitaxel, the presence of the NFL-TBS.40-63 peptide increases their uptake by glioblastoma cells in culture as evaluated by FACS analysis, and thus reduces their proliferation. Finally, when such nanocapsules were injected in mice bearing a glioma tumour, they are preferentially targeted to the tumour and reduce its progression. These results show that nanocapsules functionalized with the NFL-TBS.40-63 peptide represent a powerful drug-carrier system for glioma targeted treatment.

Axon cytoskeleton proteins specifically modulate oligodendrocyte growth and differentiation in vitro

In multiple sclerosis (MS) remyelination by oligodendrocytes (OL) is incomplete, and it is associated with a decrease in axonal neurofilaments (NF) and tubulin (TUB). To determine whether these proteins could participate directly in MS remyelination failure, or indirectly through proteins that are co-associated, we have analysed their effects in pure OL cultures. Rat brain NF fractions, recovered by successive centrifugations increase either OL progenitor (OLP) proliferation (2nd pellet, P2), or only their maturation (P5), whereas albumin, liver and skin proteins, as well as recombinant GFAP or purified actin were ineffective. NF (P2) copurify mainly with TUB, as well as with other proteins, like MAPs, Tau, spectrin β2, and synapsin 2. These purified, or recombinant, proteins increased OLP proliferation without delaying their maturation, and appeared responsible for the proliferation observed with P2 fractions. Among putative signaling pathways mediating these effects Fyn kinase was not involved. Whereas NF did not alter the growth of cultured astrocytes, the NF associated proteins enhanced their proliferation. This suggests that NF and their associated proteins exert specific effects on OL development, broadening the field of axon-oligodendrocyte interactions. In case of axon damage in vivo, extracellular release of such axonal proteins could regulate remyelination and astrocytic gliosis.

Structure-Function Analysis of the Glioma Targeting NFL-TBS.40-63 Peptide Corresponding to the Tubulin-Binding Site on the Light Neurofilament Subunit

We previously reported that a 24 amino acid peptide (NFL-TBS.40-63) corresponding to the tubulin-binding site located on the light neurofilament subunit, selectively enters in glioblastoma cells where it disrupts their microtubule network and inhibits their proliferation. Here, we analyzed the structure-function relationships using an alanine-scanning strategy, in order to identify residues essential for these biological activities. We showed that the majority of modified peptides present a decreased or total loss to penetrate in these cells, or to alter microtubules. Correspondingly, circular dichroism measurements showed that this peptide forms either β-sheet or α-helix structures according to the solvent and that alanine substitution modified or destabilized the structure, in relation with changes in the biological activities. Moreover, substitution of serine residues by phosphoserine or aspartic acid concomitantly decreased the cell penetrating activity and the structure stability. These results indicate the importance of structure for the activities, including selectivity to glioblastoma cells of this peptide, and its regulation by phosphorylation.

Abstract 2791: Disruption of EB1 comets from microtubule “plus” ends and induction of microtubule catastrophes are intrinsic mechanisms of patupilone-induced inhibition of glioblastoma cell migration

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Glioblastoma are the most frequent primary tumors of central nervous system. They are very aggressive, highly angiogenic and associated with very bad prognostic. One of the main biological features of a glioblastoma is the local invasion of its constituent neoplastic cells into the surrounding brain tissue. This invasive behavior represents a major obstacle to an effective treatment of intrinsic brain tumors. Therefore, it seems very important to target glioblastoma cell migration. Although many of the genetic alterations that deregulate the cell processes of growth and death, and are involved in tumor initiation have been elucidated in recent years, less progress has been done to understand the complex processes of migration. As a consequence, no specific therapeutic strategy against tumor cell migration is currently used to manage glioblastoma invasion. The Epothilones are a new class of microtubule (MT) stabilizing agents, which exert their cytotoxic activity by suppressing microtubule dynamics and that have strong anti-cancer activity both in vitro and in vivo. This drug family is active against cancer cells resistant to paclitaxel or to cells developing resistance to taxanes. Among the Epothilones, Patupilone (Epothilone B, EPO 906) is able to cross the blood-brain barrier since its activity is fully P-gp independent. Moreover, patupilone specifically targets αIII-tubulin, which is over expressed in glioblastomas. Here, we investigated the anti-migratory potential of patupilone through its effects on MT dynamics in glioblastoma cells. At non-cytotoxic concentrations, Patupilone inhibited glioblastoma cell migration, as shown by transwell cell migration, random motility and spheroids assay. This was associated with EB1 and related MT “plus” end tracking proteins comet shortening; and with MT catastrophes induction, without modification of any other MT dynamic instability parameters, including the microtubule growth rate. Such effect on MT catastrophes led to the disruption of MT stabilization at the leading edge. Evaluation of the effect of patupilone on EB comets and MT dynamics in a reconstituted in vitro system revealed that MT stabilizing drugs intrinsically induce MT catastrophes and disrupt EB comets at MT “plus” end. We have demonstrated that patupilone antagonizes glioblastoma cell migration by a new mechanism i.e. disruption of EB1 comets and induction of MT catastrophes distinct from its cytotoxic main mechanism i.e suppression of microtubule dynamic instability. Altogether, our results suggest that EB proteins may represent new potential target for anti-cancer therapy in highly invasive tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2791. doi:1538-7445.AM2012-2791