ENGRAILED-1 transcription factor has a paracrine neurotrophic activity on adult spinal α-motoneurons.

Several homeoprotein transcription factors transfer between cells and regulate gene expression, protein translation, and chromatin organization in recipient cells. ENGRAILED-1 is one such homeoprotein expressed in spinal V1 interneurons that synapse on α-motoneurons. Neutralizing extracellular ENGRAILED-1 by expressing a secreted single-chain antibody blocks its capture by spinal motoneurons resulting in α-motoneuron loss and limb weakness. A similar but stronger phenotype is observed in the Engrailed-1 heterozygote mouse, confirming that ENGRAILED-1 exerts a paracrine neurotrophic activity on spinal cord α-motoneurons. Intrathecal injection of ENGRAILED-1 leads to its specific internalization by spinal motoneurons and has long-lasting protective effects against neurodegeneration and weakness. Midbrain dopaminergic neurons express Engrailed-1 and, similarly to spinal cord α-motoneurons, degenerate in the heterozygote. We identify genes expressed in spinal cord motoneurons whose expression changes in mouse Engrailed-1 heterozygote midbrain neurons. Among these, p62/SQSTM1 shows increased expression during aging in spinal cord motoneurons in the Engrailed-1 heterozygote and upon extracellular ENGRAILED-1 neutralization. We conclude that ENGRAILED-1 might regulate motoneuron aging and has non-cell-autonomous neurotrophic activity.

Bidirectional transfer of homeoprotein EN2 across the plasma membrane requires PIP2.

Homeoproteins are a class of transcription factors sharing the unexpected property of intercellular trafficking that confers to homeoproteins a paracrine mode of action. Homeoprotein paracrine action participates in the control of patterning processes, including axonal guidance, brain plasticity and boundary formation. Internalization and secretion, the two steps of intercellular transfer, rely on unconventional mechanisms, but the cellular mechanisms at stake still need to be fully characterized. Thanks to the design of new quantitative and sensitive assays dedicated to the study of homeoprotein transfer within HeLa cells in culture, we demonstrate a core role of phosphatidylinositol (4,5)-bisphosphate (PIP2) together with cholesterol in the translocation of the homeobox protein engrailed-2 (EN2) across the plasma membrane. By using drug and enzyme treatments, we show that both secretion and internalization are regulated according to PIP2 levels. The requirement for PIP2 and cholesterol in EN2 trafficking correlates with their selective affinity for this protein in artificial bilayers, which is drastically decreased in a paracrine-deficient mutant of EN2. We propose that the bidirectional plasma membrane translocation events that occur during homeoprotein secretion and internalization are parts of a common process.

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.

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.

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.

Choroid plexus APP regulates adult brain proliferation and animal behavior.

Elevated amyloid precursor protein (APP) expression in the choroid plexus suggests an important role for extracellular APP metabolites such as sAPPα in cerebrospinal fluid. Despite widespread App brain expression, we hypothesized that specifically targeting choroid plexus expression could alter animal physiology. Through various genetic and viral approaches in the adult mouse, we show that choroid plexus APP levels significantly impact proliferation in both subventricular zone and hippocampus dentate gyrus neurogenic niches. Given the role of Aß peptides in Alzheimer disease pathogenesis, we also tested whether favoring the production of Aß in choroid plexus could negatively affect niche functions. After AAV5-mediated long-term expression of human mutated APP specifically in the choroid plexus of adult wild-type mice, we observe reduced niche proliferation, reduced hippocampus APP expression, behavioral defects in reversal learning, and deficits in hippocampal long-term potentiation. Our findings highlight the unique role played by the choroid plexus in regulating brain function and suggest that targeting APP in choroid plexus may provide a means to improve hippocampus function and alleviate disease-related burdens.

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 peptides) 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 thiol-containing 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-pyridinesulfenyl moiety) was introduced in its sequence.

Efficient CPP-mediated Cre protein delivery to developing and adult CNS tissues.

BACKGROUND: Understanding and manipulating gene function in physiological conditions is a major objective for both fundamental and applied research. In contrast to other experimental settings, which use either purely genetic or gene delivery (viral or non-viral) strategies, we report here a strategy based on direct protein delivery to central nervous system (CNS) tissues. We fused Cre recombinase with cell-penetrating peptides and analyzed the intracellular biological activity of the resulting chimerical proteins when delivered into cells endowed with Cre-mediated reporter gene expression. RESULTS: We show that active Cre enzymatic conjugates are readily internalized and exert their enzymatic activity in the nucleus of adherent cultured cells. We then evaluated this strategy in organotypic cultures of neural tissue explants derived from reporter mice carrying reporter "floxed" alleles. The efficacy of two protocols was compared on explants, either by direct addition of an overlying drop of protein conjugate or by implantation of conjugate-coated beads. In both cases, delivery of Cre recombinase resulted in genomic recombination that, with the bead protocol, was restricted to discrete areas of embryonic and adult neural tissues. Furthermore, delivery to adult brain tissue resulted in the transduction of mature postmitotic populations of neurons. CONCLUSION: We provide tools for the spatially restricted genetic modification of cells in explant culture. This strategy allows to study lineage, migration, differentiation and death of neural cells. As a proof-of-concept applied to CNS tissue, direct delivery of Cre recombinase enabled the selective elimination of an interneuron subpopulation of the spinal cord, thereby providing a model to study early events of neurodegenerative processes. Thus our work opens new perspectives for both fundamental and applied cell targeting protocols using proteic cargoes which need to retain full bioactivity upon internalisation, as illustrated here with the oligomeric Cre recombinase.

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.

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 non-permeability 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 most 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.

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.

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.

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.

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.