Edmond Dupont

Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone.

The amyloid precursor protein (APP) is a type I transmembrane protein of unknown physiological function. Its soluble secreted form (sAPP) shows similarities with growth factors and increases the in vitro proliferation of embryonic neural stem cells. As neurogenesis is an ongoing process in the adult mammalian brain, we have investigated a role for sAPP in adult neurogenesis. We show that the subventricular zone (SVZ) of the lateral ventricle, the largest neurogenic area of the adult brain, is a major sAPP binding site and that binding occurs on progenitor cells expressing the EGF receptor. These EGF-responsive cells can be cultured as neurospheres (NS). In vitro, EGF provokes soluble APP (sAPP) secretion by NS and anti-APP antibodies antagonize the EGF-induced NS proliferation. In vivo, sAPP infusions increase the number of EGF-responsive progenitors through their increased proliferation. Conversely, blocking sAPP secretion or downregulating APP synthesis decreases the proliferation of EGF-responsive cells, which leads to a reduction of the pool of progenitors. These results reveal a new function for sAPP as a regulator of SVZ progenitor proliferation in the adult central nervous system.

Cell-surface thiols affect cell entry of disulfide-conjugated peptides

Cell‐penetrating peptides (CPPs) can cross the cell membrane and are widely used to deliver bioactive cargoes inside cells. The cargo and the CPP are often conjugated through a disulfide bridge with the common acceptation that this linker is stable in the extracellular biological medium and should not perturb the internalization process. However, with the use of thiol‐specific reagents combined with mass spectrometry (as a quantitative method to measure intracellular concentrations of peptides) and confocal microscopy (as a qualitative method to visualize internalized pep‐tides) analyses, we could show that, depending on the peptide sequence, thiol/disulfide exchange reactions could happen at the cell surface. These exchange reactions lead to the reduction of disulfide conjugates. In addition, it was observed that not only disulfide‐ but also thiol‐containing peptides could cross‐react with cell‐surface thiols. The peptides cross‐linked by thiolcontaining membrane proteins were either trapped in the membrane or further internalized. Therefore, a new route of cellular uptake was unveiled that is not restricted to CPPs: a protein kinase C peptide inhibitor that is not cell permeant could cross cell membranes when an activated cysteine (with a 3‐nitro‐2‐pyridine‐sulfenyl moiety) was introduced in its sequence.— Aubry, S., Burlina, F., Dupont, E., Delaroche, D., Joliot, A., Lavielle, S., Chassaing, G., Sagan, S. Cell‐surface thiols affect cell entry of disulfide‐conjugated peptides. FASEB J. 23, 2956–2967 (2009). www.fasebj.org

Identification of a Signal Peptide for Unconventional Secretion

Homeoproteins are a class of transcription factors defined by the structure of their DNA-binding domain, the homeodomain. In addition to their nuclear cell-autonomous activities, homeoproteins transfer between cells, thanks to two separate steps of secretion and internalization, which both rely on unconventional mechanisms. Internalization is driven by the third helix of the homeodomain (Penetratin) through a non-vesicular and endocytosis-independent mechanism. In contrast, homeoprotein secretion involves vesicular compartments and requires the presence of a sequence of 11 amino acids (Sec sequence) spanning between the second and third helix of the homeodomain. In this study, we report that the SecPen polypeptide, which combines the two identified domains, Penetratin and Sec, bears all of the necessary information to go in and out of cells. We have analyzed key mechanisms and demonstrated that this peptide can efficiently cross a tight junction epithelium.

Penetratin Story: An Overview

Cell-penetrating peptides are short, often hydrophilic peptides that get access to the intracellular milieu. They have aroused great interest both in academic and applied research. First, cellular internalization of CPPs often involves the crossing of a biological membrane (plasma or vesicular), thus challenging the view of the nonpermeability of these structures to large hydrophilic molecules. Secondly, CPPs can drive the internalization of hydrophilic cargoes into cells, a rate-limiting step in the development of many therapeutic substances. Interestingly, the two mostly used CPPs, TAT and Penetratin peptides, are derived from natural proteins, HIV Tat and Antennapedia homeoprotein, respectively. The identification of the Penetratin peptide, summarized in this review, is intimately linked to the study of its parental natural protein.

Homeoprotein Signaling in Development, Health, and Disease: A Shaking of Dogmas Offers Challenges and Promises from Bench to Bed

Homeoproteins constitute a major class of transcription factors active throughout development and in adulthood. Their membrane transduction properties were discovered over 20 years ago, opening an original field of research in the domain of vector peptides and signal transduction. In early development, homeoprotein transfer participates in tissue patterning, cell/axon guidance, and migration. In the axon guidance model, homeoproteins exert their non–cell autonomous activity through the regulation of translation, in particular, that of nuclear-transcribed mitochondrial mRNAs. An important aspect of these studies on patterning and migration is that homeoproteins sensitize the cells to the action of other growth factors, thus cooperating with established signaling pathways. The role of homeoprotein signaling at later developmental stages is also of interest. In particular, the transfer of homeoprotein Otx2 into parvalbumin-expressing inhibitory neurons (PV-cells) in the visual cortex regulates cortical plasticity. The molecular deciphering of the interaction of Otx2 with binding sites at the surface of PV-cells has allowed the development of a specific Otx2 antagonist that reopens plasticity in the adult cortex and cures mice from experimental amblyopia, a neurodevelopmental disease. Finally, the use of homeoproteins as therapeutic proteins in mouse models of glaucoma and Parkinson disease is reviewed. In the latter case, engrailed homeoproteins protect mesencephalic dopaminergic neurons by increasing the local translation of complex I mitochondrial mRNAs. In conclusion, this review synthesizes 20 years of work on the fundamental and potentially translational aspects of homeoprotein signaling.

Modifications in the chemical structure of Trojan carriers: impact on cargo delivery

Carriers with linear or dendrimeric structures displaying different functional groups were synthesized and their delivery properties were studied.

Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development

It is now becoming evident that hydrogen peroxide (H2O2), which is constantly produced by nearly all cells, contributes to bona fide physiological processes. However, little is known regarding the distribution and functions of H2O2 during embryonic development. To address this question, we used a dedicated genetic sensor and revealed a highly dynamic spatio-temporal pattern of H2O2 levels during zebrafish morphogenesis. The highest H2O2 levels are observed during somitogenesis and organogenesis, and these levels gradually decrease in the mature tissues. Biochemical and pharmacological approaches revealed that H2O2 distribution is mainly controlled by its enzymatic degradation. Here we show that H2O2 is enriched in different regions of the developing brain and demonstrate that it participates to axonal guidance. Retinal ganglion cell axonal projections are impaired upon H2O2 depletion and this defect is rescued by H2O2 or ectopic activation of the Hedgehog pathway. We further show that ex vivo, H2O2 directly modifies Hedgehog secretion. We propose that physiological levels of H2O2 regulate RGCs axonal growth through the modulation of Hedgehog pathway.

Homeoproteins and Homeoprotein-derived Peptides: Going in and Out

Since the initial evidence that antennapedia homeobox can cross cell membranes and internalize into cells, numerous peptides with similar translocation properties have been described. These peptides are referred to as cell-penetrating peptides (CPPs) or protein-transduction domains (PTDs). Reviews on reported CPP sequences have been recently published, together with reviews on their mechanisms of internalization. In this review, we will focus on natural homeoproteins and homeoprotein-derived peptides and describe results that have been obtained among different laboratories to unravel the different pathways by which these molecules reach the cell cytosol and nucleus or transfer from one cell to another. Using homeoproteins as a paradigm, we will also summarize recent evidences of the physiological functions of endogenous protein translocation.

Nerves, H2O2 and Shh: Three players in the game of regeneration

The tight control of reactive oxygen species (ROS) levels is required during regeneration. H2O2 in particular assumes clear signalling functions at different steps in this process. Injured nerves induce high levels of H2O2 through the activation of the Hedgehog (Shh) pathway, providing an environment that promotes cell plasticity, progenitor recruitment and blastema formation. In turn, high H2O2 levels contribute to growing axon attraction. Once re-innervation is completed, nerves subsequently downregulate H2O2 levels to their original state. A similar regulatory loop between H2O2 levels and nerves also exists during development. This suggests that redox signalling is a major actor in cell plasticity.

Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain

Homeoproteins of the Engrailed family are involved in the patterning of mesencephalic boundaries through a mechanism classically ascribed to their transcriptional functions. In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation. Using time-controlled activation of Engrailed combined with tools that block its transfer, we show that the positioning of the diencephalic-mesencephalic boundary (DMB) requires Engrailed paracrine activity. Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning. In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway. SUMMARY: The paracrine activity of Engrailed, together with its PBX-interacting hexapeptide motif, is involved in boundary formation during brain development in zebrafish.