Biomedical subjects

A Prochiantz

Publications and source records attributed to A Prochiantz.

At least 55 records · Page 3Linked to original sources

PubMed1994

The third helix of the Antennapedia homeodomain translocates through biological membranes.

The 60-amino acid long homeodomain of Antennapedia crosses biological membranes by an energy-independent mechanism, a phenomenon abolished by directed mutagenesis within the polypeptide C-terminal region. This finding led us to study the internalization of several chemically synthesized peptides derived from the third helix of the homeodomain. We report here that a polypeptide of 16 amino acids in length corresponding to the third helix of the homeodomain deleted of its N-terminal glutamate is still capable of translocating through the membrane. A longer peptide of 20 amino acids also translocates, whereas shorter peptides (15 amino acids) are not internalized by the cells. As is also the case for the entire homeodomain, the 20- and 16-amino acid long peptides are internalized at 4 degrees C, suggesting an energy-independent mechanism of translocation not involving classical endocytosis. The two translocated peptides can be recovered, intact, within the cells, strongly suggesting that they are not targeted to the lysosomal compartment. Finally, substitution of two tryptophans by two phenylalanines strongly diminishes translocation, raising the possibility that the internalization of the third helix is not solely based on its general hydrophobicity.

Amino Acid Sequence↗

PubMed1994

Defined glycosaminoglycan motifs have opposite effects on neuronal polarity in vitro.

We previously reported that heparan sulfates enhance axonal outgrowth and inhibit dendrite elongation, whereas dermatan sulfates favor the development of both axons and dendrites. The present study focuses on the activity of small synthetic heparan or dermatan sulfate-like compounds. We found three heparan sulfate-like and three dermatan sulfate-like sugars that mimic the morphological effects of the high-molecular-weight natural glycosaminoglycans. Indeed, heparan sulfate-like compounds enhance axonal maturation and inhibit dendrite growth whereas the active sugars from the dermatan sulfate series act primarily on the elongation of cortical dendrites. The effect of dermatan sulfate-like sugars on cortical dendrite growth is only observed on the subpopulation of neurons with an established axon. We also studied the effects of the synthetic sugars on motoneurons. We found that the response of motoneurons to heparan sulfate-like compounds is indistinguishable from that of cortical neurons but that dermatan sulfate-like sugars do not enhance the development of motoneuron dendrites. The distinct effects of the two types of sugars and the fact that their activity only requires a short period of contact with the cells suggest the existence of specific binding sites for dermatan-like and heparan-like compounds. This possibility is reinforced by the fact that the binding and internalization of natural heparin fragments by neurons in culture is competitively inhibited by synthetic heparan sulfate-like derivatives, but not by dermatan sulfate-like derivatives.

Animals↗

PubMed1994

Neurotrophic activity of a homeobox peptide.

Homeoproteins are well known for their role in defining the shape of organs during early development. The late expression of some homeogenes in the nervous system suggests that they might have other, additional functions, possibly in neurite growth and target recognition. The 60 amino acid-long peptide corresponding to the homeobox of Antennapedia (pAntp) translocates through the membrane of neurons in culture and reaches their nuclei. This process is followed by an enhanced morphological differentiation of the neurons. Internalization by neurons is four-fold that observed with fibroplasts. This difference is abolished upon treatment with Endo-N which specifically cleaves alpha,2-8 bonds in polysialic acid. To understand the mode of action of the peptide, we constructed three mutants modified in their capacity to specifically bind promoters and/or to translocate through the cell membrane. The biological properties of the mutants demonstrate that the neurotrophic action of pAntp requires its internalization and integrity of its specific DNA-binding capacity.

Animals↗

PubMed1994

Rab3A and Rab3B carboxy-terminal peptides are both potent and specific inhibitors of prolactin release by rat cultured anterior pituitary cells.

Chimeric polypeptides composed of the homeodomain of Antennapedia and of the C-terminus of several low molecular weight GTP-binding proteins of the rab family have been found to translocate through the membrane of cells in culture and to accumulate in the cytoplasm and nucleus. We have used these chimeric peptides to study, in intact endocrine cells, a putative role for the C-terminal domain of rab proteins in the exocytotic process. We show that the internalization of 33- and 32-amino acid polypeptides corresponding to the C-terminal domains of rab3A and rab3B blocks calcium-triggered PRL release from adult rat anterior pituitary cells maintained in primary culture. This effect is specific to rab3 since it is not observed after internalization of either rab1 or rab2 C-terminal peptides. In addition, we demonstrate that this inhibition does not require the geranylgeranylation of the internalized C-termini. As rab3B normally shows a permissive effect on exocytosis in PRL-secreting cells, we suggest that the C-terminal domains of rab3A and rab3B contain structural elements that compete with endogenous rab3 necessary for calcium-induced exocytosis.

Amino Acid Sequence↗

PubMed1994

Region-specific neuroastroglial interactions in neuronal morphogenesis and polarity: from homeogenic induction to cellular cytomechanics.

Because of several studies on neuronal differentiation, it has been possible to produce a list of adhesion molecules, cytoskeletal elements, and transacting factors that participate in the determination of neuronal shape and polarity. Most of these proteins are ubiquitous, and this lack of regional specificity is unlikely to be compensated for by editing mechanisms, such as alternative splicing and post-translational modifications. In this context it is quite difficult to understand the regional specificity of neuroneuronal or neuroastroglial interactions. For this reason, we are interested in the study of homeoproteins, a class of transcription factors endowed with regional specificity, which regulate the synthesis of several morphoregulatory molecules, including cell and substrate adhesion molecules. We propose a model in which specific neuroastroglial and neuroneuronal interactions result from the homeoprotein-dependent regulation of the quantities and of the topological organization of such ubiquitous morphoregulatory molecules. Moreover, we extend this model by proposing that some homeoprotein isoforms could act not only according to a classic intracrine mode, but also through a paracrine mechanism.

Animals↗

PubMed1994

Amyloid precursor protein in cortical neurons: coexistence of two pools differentially distributed in axons and dendrites and association with cytoskeleton.

Embryonic cortical neurons in culture contain transmembrane amyloid precursor protein (APP) capable of associating with the detergent-insoluble cytoskeleton through interactions requiring the presence of its C-terminal. These transmembrane APPs are not detectable at the surface of living cells. When neurons are fixed with paraformaldehyde alone, APP is mainly visualized close to the membrane of the axon and cell body of 40% of neurons, with virtually no dendritic staining. Membrane permeabilization with detergent or methanol extends APP immunostaining to 100% of the cells and to all compartments, including the dendrites. Taken together, these results suggest that APP in embryonic neurons is present in two compartments, one more readily detectable in some axons and cell bodies and the other distributed throughout all neurons. The axonal and somatic pool of APP detectable after paraformaldehyde fixation alone is highly and rapidly augmented after exposure to calcium ionophores. We propose that calcium entry increases the amount of axonal APP close to the cell surface, but that the stabilization of the protein at the cell surface and its subsequent secretion require further physiological stimuli.

Amino Acid Sequence↗

PubMed1993

Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties.

In previous reports we have demonstrated that the 60-aa peptide corresponding to the homeodomain of the Drosophila protein Antennapedia (pAntp) translocates through the membrane of neurons in culture, accumulates in neuronal nuclei, and promotes neurite growth. To analyze the importance of specific pAntp DNA-binding properties in this phenomenon we have constructed three mutant versions of pAntp that differ in their ability to translocate through the membrane and to bind specifically the cognate sequence for homeodomains present in the promoter of HoxA5. We demonstrate that removing two hydrophobic residues of the third helix inhibits pAntp internalization and suppresses its neurotrophic activity. We also show that pAntp neurotrophic activity is lost when mutations are introduced in positions preserving its penetration and nuclear accumulation but abolishing its capacity to bind specifically the cognate DNA-binding motif for homeoproteins. Our results strongly suggest that pAntp neurotrophicity requires both its internalization and its specific binding to homeobox cognate sequences. We propose that homeoproteins might regulate important events in the morphological differentiation of the postmitotic neuron.

Animals↗

PubMed1993

Astrocyte-regulated synaptogenesis: an in vitro ultrastructural study.

Striatal neurons from E15 rat embryos were dissociated, plated at low cell density on polyornithine or on astrocyte monolayers derived from the striatum (homotopic) or mesencephalon (heterotopic), and cultured in a chemically defined medium. Dendrites developing in homotopic co-cultures could reach a state of maturation allowing the establishment of synapses with axons from mesencephalic explants. This culture system thus partially reproduces the in vivo conditions in which striatal neurons developing in an homotopic glial environment can serve as synaptic targets for afferent mesencephalic axons.

Animals↗

PubMed1993

Antennapedia homeobox peptide enhances growth and branching of embryonic chicken motoneurons in vitro.

Spinal motoneuron development is regulated by a variety of intrinsic and extrinsic factors. Among these, a possible role for homeoproteins is suggested by their expression in the motoneuron at relatively late stages. To investigate their possible involvement in motoneuron growth, we adapted a novel technique recently developed in this laboratory, based on the ability of the 60 amino acid-long homeobox of Antennapedia (pAntp) to translocate through the neuronal membrane and to accumulate in the nucleus (Joliot, A. H., C. Pernelle, H. Deagostini-Bazin, and A. Prochiantz. 1991. Proc. Natl. Acad. Sci. USA. 88:1864-1868; Joliot, A. H., A. Triller, M. Volovitch, C. Pernelle, and A. Prochiantz. 1991. New Biol. 3:1121-1134). Motoneurons from E5 chicken spinal cord were incubated with pAntp, purified by panning on SC1 antibody and plated on polyornithine/laminin substrata without further addition of pAntp. After 24 h, neurite outgrowth was already extensive in controls. In cultures of motoneurons that had been preincubated with 10(-7) M pAntp, neurite length was doubled; a similar effect was obtained using postnatal muscle extracts. Morphological analysis using a neurofilament marker specific for axons indicated that the homeobox peptide enhances primarily axonal elongation and branching. To test the hypothesis that the biological activity of pAntp involves its specific attachment to cognate homeobox binding sites present in the genome, we generated a mutant of pAntp called pAntp40P2, that was still able to translocate through the motoneuron membrane and to reach the nucleus, but had lost the specific DNA-binding properties of the wild-type peptide. Preincubation of pAntp40P2 with purified motoneurons failed to increase neurite outgrowth. This finding raises the possibility that motoneuron growth is controlled by homeobox proteins.

Animals↗

PubMed1993

Specific responses of axons and dendrites to cytoskeleton perturbations: an in vitro study.

Several factors can influence the development of axons and dendrites in vitro. Some of these factors modify the adhesion of neurons to their substratum. We have previously shown that the threshold of neuron-substratum adhesion necessary for initiation and elongation of dendrites is higher than that required for axonal growth. To explain this difference we propose that, in order to antagonize actin-driven surface tension, axons primarily rely on the compression forces of microtubules whereas dendrites rely on adhesion. This model was tested by seeding the cells in conditions allowing the development either of axons or of axons and dendrites, then adding cytochalasin B or nocodazole 1 hour or 24 hours after plating. The addition of cytochalasin B, which depolymerizes actin filaments and thus reduces actin-tensile forces, increases the length of both axons and dendrites, indicating that both axons and dendrites have to antagonize surface tension in order to elongate. The addition of nocodazole, which acts primarily on microtubules, slightly reduces dendrite elongation and totally abolishes axonal growth. Similar results are obtained when the drugs are added 1 or 24 hours after plating, suggesting that the same mechanisms are at work both in initiation and in elongation. Finally, we find that in the presence of cytochalasin B axons adopt a curly morphology, a fact that could be explained by the importance of tensile forces in antagonizing the asymmetry created by polarized microtubules presenting a uniform minus/plus orientation.

Actins↗

PubMed1993

[Neurotrophic activity of homeopeptide].

The sixty aminoacid-long peptide corresponding to the homeobox of Antennapedia (pAntp) translocates through the membrane of neurons in culture and reaches their nuclei. This process is followed by an enhanced morphological differentiation of the neurons. Internalization by neurons is 4-fold that observed with fibroblasts. This difference is abolished upon treatment Endo-N which specifically cleaves alpha, 2-8 bounds in polysialic acid (PSA). To understand the mode of action of the peptide, we constructed three mutants modified in their capacity to specifically bind promoters and/or to translocate through the cell membrane. The biological properties of the mutants demonstrate that the neurotrophic action of pAntp requires its internalization and the integrity of its specific DNA-binding capacity.

Animals↗

PubMed1993

Control of neuronal morphogenesis by homeoproteins: consequences for the making of neuronal networks.

To test whether homeoproteins can act as genetic regulators in the processes of neurite growth, branching, guidance, and connectivity, the 60 amino acid homeodomain of Antennapedia was introduced in embryonic neurons in primary culture. It was hoped that this homeopeptide would bind to specific promoters and thus behave as a competitive inhibitor of endogenous homeoproteins. The introduction of the homeodomain in the nerve cells was made easy by its unexpected capability to translocate through the membranes and to accumulate within the nuclei. The presence of the homeodomain within the cells correlated with an increase in neurite growth and branching. The absence of activity of mutant peptides, still internalized but unable to bind with high affinity to homeoprotein cognate binding sites, strongly suggested that endogenous homeoproteins modulate neurite outgrowth and branching. Moreover, the efficient internalization of the homeobox peptide by live cells in culture raises the possibility that, in addition to their well-established role as cell-autonomous transcription factors, some homeoproteins may also exert paracrine functions. We examine how these hypotheses could modify our current views on the establishment and plasticity of neuronal networks.

Animals↗

PubMed1993

Neurotrophic activity of an homeobox peptide.

The sixty aminoacid-long peptide corresponding to the homeobox of Antennapedia (pAntp) translocates through the membrane of neurons in culture and reaches their nuclei. This process is followed by an enhanced morphological differentiation of the neurons. Internalization by neurons is 4-fold that observed with fibroblasts. This difference is abolished upon treatment with Endo-N which specifically cleaves alpha, 2-8 bounds in polysialic acid (PSA). To understand the mode of action of the peptide, the authors constructed three mutants modified in their capacity to specifically bind promoters and/or to translocate through the cell membrane. The biological properties of the mutants demonstrate that the neurotrophic action of pAntp requires its internalization and the integrity of its specific DNA-binding capacity.

Animals↗

PubMed1993

Astrocyte-regulated GABA-ergic striatal neurons development: an in vitro ultrastructural study.

Striatal neurons from E15 rat embryos were dissociated, plated at low cell density on polyornithine or on astrocyte monolayers derived from the striatum (homotopic) or mesencephalon (heterotopic), and cultured in a chemically defined medium. After 2 to 10 days neurons could be divided in 3 classes according to their cell body diameter: small, medium or large. The percentage of small neurons which was very high 60% for GABAergic neurons on polyornithine after 2 days in vitro was reduced to 35% on mesencephalic astrocytes and to less than 20% on striatal astrocytes. The decrease in the number of small cells was paralleled by an increase in the number of multipolar medium size cells whereas the percentages of bipolar medium size and large neurons remained constant (55 and 4% respectively). All results obtained with the general neuronal population were replicated with the GABAergic sub-population which accounted for more than 50% of total neuronal population. These experiments confirm the beneficial influence of homotopic astrocytes on neuronal differentiation and on dendrite growth.

Animals↗

PubMed1992

In vitro control of neuronal polarity by glycosaminoglycans.

We have studied the effects of proteoglycans (PGs) and glycosaminoglycans (GAGs) on the growth and morphology of neurons in culture. PGs from glial cells or Engelbreth-Holm-Swarm tumor cells (EHS), pure bovine kidney heparan sulfate (HS), shark cartilage type C chondroïtin sulfate (CSc) and bovine mucosa dermatan sulfate (DS) added to embryonic rat neurons strongly enhanced total neurite growth after 48 h in vitro. No trophic effects were seen when PGs treated with a mixture of glycanases were used. PGs, CSc and HS not only enhanced neurite growth but induced the appearance of a majority of neurons with a single long axon whereas, in contrast, DS increased dendrite growth. GAGs bound to the cell surface and were rapidly internalized, a feature that correlated well with the absence of neurotrophicity of GAGs previously immobilized on the culture substratum. Although the mechanisms involved in GAGs neurotrophic effects and in the separate regulation of neuronal polarity by HS and DS were not elucidated, we found that, as opposed to HS, DS was able to enhance neuronal adhesion and spreading and to maintain a high level of expression of microtubule-associated protein 2 (MAP2), a specific dendritic marker. This finding confirms and extends our previous observations on the role of adhesion in the regulation of dendrite growth.

Animals↗

PubMed1992

Antennapedia homeobox as a signal for the cellular internalization and nuclear addressing of a small exogenous peptide.

In a previous study we demonstrated that a homeobox peptide corresponding to the 60 amino acid long DNA-binding region of the Drosophila antennapedia homeo-protein was capable of crossing the plasma membrane of cells in culture. This finding has led us to investigate whether chimeric molecules encompassing the homeobox would behave in a similar manner. We demonstrate here that a peptide of 93 amino acids composed of the homeobox and of the C terminus of Rab3, a small GTP-binding protein, crosses the membrane of myoblasts, myotubes and neurons and is conveyed to their nuclei. This transport is highly efficient, is observed in all the cells present in the culture and occurs at 37 degrees C and 12 degrees C without quantitative peptide degradation. Beyond its theoretical implications for our current views on cellular interactions, this finding could have technical repercussions on the development of drugs with intracellular targets.

Animals↗

PubMed1992

[Are embryonic forms of NCAM homeobox receptors?].

In a previous study we demonstrated that the homeobox peptide pAntp was able to penetrate into rat embryonic neurons in culture thus provoking their morphological differentiation [1]. In the present work we have started to analyse the process of penetration of the homeobox peptide. As illustrated in Figure 1 pAntp migrating as a homogeneous 7 kDa band could be recovered in the nuclear fraction 2 hrs. only after its addition to cultured embryonic neurons (lane 2). Penetration and nuclear targeting were quantitatively blocked by preincubating pAntp with its cognate recognition sequence present in the promoter of Hox-1.3 (lane 3) or by incubating the cells with an antibody directed against the NCAM-specific alpha 2-8 polysialic acid (lane 4). Similar inhibitions were observed when the peptide was incubated with double stranded DNA or added to cells deprived of alpha 2-8 polysialic acid by EndoN treatment (not shown). As illustrated in Figure 2, the strong pAntp-induced neurite growth was antagonized when pAntp internalization was prevented by the EndoN removal of PSA. This effect of EndoN was not due to the enzyme itself since morphological differentiation was not inhibited if EndoN was added after pAntp penetration (Fig. 2B). Polysialic acid is composed of long chains of neuraminic acids, each pyranose ring carrying a negatively charged carboxylic group linked to carbon in position 1. NMR studies of the molecule in solution have demonstrated that the alpha 2-8 link between each pyranose ring allows specific and stable helical conformation [2].(ABSTRACT TRUNCATED AT 250 WORDS)

Antennapedia Homeodomain Protein↗

PubMed1991

Antennapedia homeobox peptide regulates neural morphogenesis.

We synthesized the 60-amino acid polypeptide corresponding to the sequence of the Drosophila antennapedia gene homeobox. This peptide (pAntp) recognized the consensus motif for binding to the promoter region of Hox-1.3. pAntp mechanically introduced into mammalian nerve cells provoked a dramatic morphological differentiation of the neuronal cultures. Moreover, pAntp directly added to already differentiated neuronal cultures penetrated the cells and further augmented their morphological differentiation. Examination of live and fixed neurons in classical and confocal fluorescence microscopy demonstrated that pAntp was captured at all regions of the nerve cells and accumulated in the nuclei. In addition, the effect of pAntp on neurite extension was blocked in the presence of the protein synthesis inhibitor cycloheximide. Thus, our results demonstrate that neurons possess an efficient uptake system for the antennapedia homeobox peptide and suggest that binding of pAntp to consensus motifs present in nerve cell nuclei influences neuronal morphogenetic programs.

Animals↗