US20120015884A1 - Polypeptides for Specific Targeting to Otx2 Target Cells - Google Patents

Polypeptides for Specific Targeting to Otx2 Target Cells Download PDF

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Publication number
US20120015884A1
US20120015884A1 US13/145,066 US201013145066A US2012015884A1 US 20120015884 A1 US20120015884 A1 US 20120015884A1 US 201013145066 A US201013145066 A US 201013145066A US 2012015884 A1 US2012015884 A1 US 2012015884A1
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Prior art keywords
otx2
polypeptide
neurons
cells
molecule
Prior art date
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Abandoned
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US13/145,066
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Inventor
Alain Prochiantz
Ariel Di Nardo
Marine Beurdeley
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Centre National de la Recherche Scientifique CNRS
Ecole Normale Superieure
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Centre National de la Recherche Scientifique CNRS
Ecole Normale Superieure
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Assigned to CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE, ECOLE NORMALE SUPERIEURE reassignment CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BEURDELEY, MARINE, DI NARDO, ARIEL, PROCHIANTZ, ALAIN
Publication of US20120015884A1 publication Critical patent/US20120015884A1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: BOSTON CHILDREN'S HOSPITAL
Assigned to NIH-DEITR reassignment NIH-DEITR CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: BOSTON CHILDREN'S HOSPITAL
Abandoned legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/164—Retinal disorders, e.g. retinopathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/168—Glaucoma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds

Definitions

  • the present invention relates to polypeptides which enable the specific targeting of a molecule of interest to homeoprotein Otx2 target cells, and in particular to retinal ganglion neurons and retinal bipolar neurons.
  • the retina is the cell sheet coating the back of the eye. It contains various types of neurons, the role of which is to be capture light energy and to convert it into a nerve signal, and also glial cells.
  • the retina comprises three main layers of neurons: photoreceptor neurons (cones and rods), bipolar neurons and ganglion neurons; other neurons, the amacrine neurons and the horizontal neurons, play a regulatory role.
  • the photoreceptor neurons react to light, and the signal that they generate is transmitted, by means of the bipolar neurons, to the ganglion neurons, the axons of which constitute the nerve fibers of the optic nerve, sending information to the brain.
  • the degeneration of retinal neurons is implicated in various retinopathies.
  • the degeneration of photoreceptor neurons is implicated in certain pathological conditions, such as pigmentary retinitis or macular degeneration.
  • pathological conditions such as glaucoma, it is mainly the ganglion neurons which are affected.
  • Otx2 (Orthodenticle homolog 2) is a homeoprotein containing a bicoid-type homeodomain (Simeone et al., Embo J, 12, 2735-47, 1993). It belongs to the Otx homeoprotein family, which plays a fundamental role in brain development during embryogenesis (Acampora et al., Prog Neurobiol, 64, 69-95, 2001; Simeone et al., Curr Opin Genet Dev, 12, 409-15, 2002).
  • Otx2 (Orthodenticle homolog 2) is a homeoprotein containing a bicoid-type homeodomain (Simeone et al., Embo J, 12, 2735-47, 1993). It belongs to the Otx homeoprotein family, which plays a fundamental role in brain development during embryogenesis (Acampora et al., Prog Neurobiol, 64, 69-95, 2001; Simeone et al., Curr Opin Genet Dev, 12, 409-15, 2002). It has also been shown that Otx2 plays a role in post-natal development of the cerebral cortex, and in particular in its plasticity.
  • Otx2 which is synthesized in the retina, is transported to the visual cortex, where it is taken up by the parvalbumin neurons of the visual cortex, thereby inducing their maturation, and leads first to the opening, and then, some time later, the closing of the critical period of plasticity (Sugiyama et al., Cell, 134, 508-20, 2008).
  • a critical period of plasticity is a phase of post-natal development represented by a time interval of limited duration (variable according to animal species and according to the sensory function involved) during which the sensory stimuli are capable of modifying the functional organization of the corresponding cortical zones.
  • Otx2 binds specifically to ganglion neurons and to bipolar neurons, and have identified the region of Otx2 that is responsible for this binding. They have also shown that this same region is also responsible for the targeting of Otx2 to the parvalbumin neurons of the visual cortex, by interacting with the perineuronal net of chondroitin sulfate proteoglycans which surrounds these neurons, and that it makes it possible, by competing with the endogenous Otx2 protein taken up by the parvalbumin neurons, to inhibit this uptake. This inhibition leads to the return of these neurons to an immature state, enabling re-opening of the critical phase of plasticity.
  • the region responsible for the targeting of Otx2 to its target cells is made up of a peptide sequence of 15 amino acids.
  • the inventors have also noted that this polypeptide, isolated, has the same binding specificity as the whole Otx2 protein.
  • a subject of the present invention is an isolated cell targeting polypeptide defined by the sequence B 1 B 2 X 1 B 3 B 4 X 2 B 5 X 3 X 4 X 5 X 6 B 6 X 7 X 8 X 9 (SEQ ID NO: 1), in which:
  • B 1 , B 2 , B 3 , B 4 , B 5 et B 6 represent independently arginine or lysine
  • X 1 and X 8 are independently asparagine or glutamine
  • X 2 represents aspartic acid or glutamic acid
  • X 3 , X 4 and X 6 represent independently threonine or serine
  • X 5 represents phenylalanine, tyrosine or tryptophan
  • X 7 represent alanine or glycine
  • X 9 represents leucine, isoleucine or valine.
  • This polypeptide in the presence of retinal cells, binds specifically to ganglion neurons and to bipolar neurons.
  • a cell targeting polypeptide in accordance with the invention is defined by the following sequence: RKQRRERTTFTRAQL (SEQ ID NO: 2).
  • the amino acids constituting a polypeptide in accordance with the invention may be natural amino acids of the L series. It is also possible to replace all or some of these amino acids with their isomers of the D series, in order to increase the stability of the polypeptide in vivo.
  • the amino acids, of the L series or of the D series may also, where appropriate, be linked according to a reverse sequence of the sequence SEQ ID NO: 1 or of the sequence SEQ ID NO: 2, which sequences are indicated above.
  • a subject of the present invention is also the use of a cell targeting polypeptide in accordance with the invention, for enabling the specific targeting of a cargo of interest to Otx2 target cells.
  • Otx2 target cells By way of examples of Otx2 target cells, mention will be made, in addition to the retinal ganglion neurons and retinal bipolar neurons already mentioned above, of the neurons coated in a perineuronal net of chondroitin sulfate proteoglycans, which includes in particular neurons expressing parvalbumin (PV neurons) located in particular in the visual cortex.
  • Other Otx2 target cells are neurons of the midbrain, in particular the dopaminergic neurons of the substantia nigra and of the ventral tegmental area, and their synaptic targets.
  • Otx2 target cells can be easily identified by means of a targeting polypeptide in accordance with the invention, for example by combining said polypeptide with a label, by bringing said labeled polypeptide into contact with a sample of a tissue or of an organ to be tested, and by detecting, in said sample, the presence or absence of cells binding said polypeptide, and in the case of the presence of cells binding said polypeptide, their localization.
  • “cargo” denotes any molecule or molecular complex that it is desired to target to a target cell.
  • the cargos that can be transported by cell targeting polypeptides in accordance with the invention may be of very varied nature: they may be chemical molecules, macromolecules, for instance proteins or nucleic acids, or particles such as liposomes, nanoparticles, or viral or virus-like particles. They may be labels intended to make it possible to detect and/or localize Otx2 target cells in a tissue or an organ, or active ingredients that it is desired to specifically target to Otx2 target cells.
  • a cell targeting polypeptide in accordance with the invention can advantageously be associated with a transducer polypeptide.
  • Transducer polypeptides are polypeptides comprising a sequence called a “transduction domain” which confers on them the ability to penetrate inside a living cell, independently of the presence of specific transporters or receptors.
  • transducer polypeptides are known per se.
  • penetratins which are polypeptides derived from the third helix of a homeodomain
  • polypeptides derived from the Tat protein of HIV1, and in particular from fragment 48-60 of said protein polyarginines
  • polypeptides derived from the VP22 protein of HSV polypeptides derived from a signal sequence conjugated to a nuclear localization sequence
  • transportans which are derived from a fusion between a portion of a neuropeptide, galanin, and a wasp venom polypeptide.
  • the subject of the present invention is also an isolated polypeptide containing a cell targeting polypeptide in accordance with the invention and a transducer polypeptide.
  • This polypeptide may be an isolated fragment of Otx2 comprising the entire homeodomain, and the 2 amino acids immediately preceding said homeodomain. This fragment may also be deleted of a part of the sequence of the homeodomain, provided that the targeting sequence in accordance with the invention and at least the third helix of the homeodomain are retained.
  • the polypeptide may also be a chimeric polypeptide associating a cell targeting polypeptide in accordance with the invention with a heterologous transducer polypeptide.
  • preferred transducer polypeptides are those of the penetratin family. It is thus possible to associate a cell targeting polypeptide in accordance with the invention with a homeodomain fragment of a homeoprotein other than Otx2, comprising at least the third helix of said homeodomain, or else with penetratin derivatives such as those described, for example, in PCT applications WO 00/01417 or WO 00/29427.
  • compositions comprising a cell targeting polypeptide in accordance with the invention, optionally associated with a transducer polypeptide, bonded to a cargo.
  • the bonding between the cell targeting polypeptide in accordance with the invention and the cargo can be carried out in various ways, known per se, according in particular to the nature of the cargo concerned, and the envisaged modes of use.
  • the cell targeting polypeptide (optionally fused with a transducer polypeptide) and the cargo will be covalently associated, where appropriate by means of a spacer arm, for example a peptide linker. They may also be noncovalently associated, by means of ionic or hydrophobic interactions; in this case, the targeting polypeptide may be bonded to a molecule capable of noncovalently bonding to the cargo.
  • This molecule may in particular be a transducer polypeptide such as a penetratin, capable of bonding, via hydrophobic interactions, to a cargo having one or more hydrophobic domains, as described in PCT application WO 04/069279.
  • composition in accordance with the invention is in the form of a chimeric polypeptide, comprising a cell targeting polypeptide in accordance with the invention, bonded with one or more polypeptide sequences constituting the cargo, and optionally with a transducer polypeptide.
  • the order in which the cell targeting polypeptide, the transducer polypeptide and the polypeptide sequences constituting the cargo are arranged is not essential.
  • chimeric polypeptides comprising a cell targeting polypeptide in accordance with the invention, a transducer polypeptide, one or more transcription-regulating sequences and/or one or more translation-regulating sequences.
  • the term “chimeric polypeptide” is used herein in its usual sense, to denote polypeptides associating sequences of different origins, which therefore excludes natural Otx2 proteins.
  • chimeric polypeptides in accordance with the invention can be obtained by various methods that are well-known per se, in particular by peptide synthesis, or by conventional generic engineering techniques.
  • Chimeric polypeptides in accordance with the invention comprising a cell targeting polypeptide, a transducer polypeptide, one or more transcription-regulating sequences and/or one or more translation-regulating sequences can be used in the same applications as the natural Otx2 protein, and in particular for increasing the survival of Otx2 target cells. They can thus be used in particular for preventing or treating the degeneration of retinal ganglion neurons and/or retinal bipolar neurons, which are involved in particular in glaucoma, and also in various optical, genetic or vascular neuropathies, for example pigmentary retinitis or optic nerve damage. They can also be used in the context of the treatment of certain neurodegenerative diseases (such as, for example, Alzheimer's disease, multiple sclerosis or Parkinson's disease). Generally, these chimeric polypeptides may be used in said applications according to the same modes as those described for Otx2 in PCT application WO 2009/106767.
  • bringing said chimeric polypeptide into contact with the target cells is carried out at a concentration of said chimeric polypeptide of from 0.5 to 10 nM, advantageously from 1 to 5 nM, and particularly advantageously from 1.5 to 3 nM.
  • chimeric polypeptide In vitro, all that is needed is to add said chimeric polypeptide to the neuron culture medium.
  • it can be administered via various routes, locally, in particular by injection or infusion into the vitreous humor or into the infraorbital space, or in the form of an eyewash or of an ophthalmic ointment. It can also be administered using a controlled-release device, for example in the form of an intraocular implant. Where appropriate, it can be administered systemically, for example by intravenous injection.
  • chimeric polypeptide to be administered in vivo in order to obtain the desired concentration in contact with the target cells can be readily be determined and adjusted by those skilled in the art depending, in particular, on the methods of administration envisaged.
  • This contact can also be brought about by placing the target cells in the presence of cells that have been transformed so as to express or overexpress, and secrete, said chimeric polypeptide. In vitro, this can be carried out by coculturing these transformed cells with the cells. In vivo, cells transformed so as to express or overexpress, and secrete, said chimeric polypeptide can, for example, be grafted into the retina.
  • Cell targeting polypeptides in accordance with the invention can also be used for inhibiting the binding of Otx2 to its target cells, and in particular to parvalbumin neurons coated in a perineuronal net of chondroitin sulfate proteoglycans, so as to make it possible to restore their plasticity. They can thus be used for the treatment of diseases resulting from a defective development, during the critical period of plasticity, of a region of the brain containing the target cells concerned. By way of example, they can be used in the context of the treatment of amblyopia, or in the context of the treatment of neurological or psychiatric diseases such as anxiety disorders, post-traumatic stress syndrome, and also manic-depressive psychosis or schizophrenia. They can also be used in the context of restoring physiological and morphological plasticity in pathological conditions or strokes which lead to the loss of neurons.
  • the cell targeting polypeptide in accordance with the invention will be used in such a way as to obtain, on contact with said target cells, a concentration of said polypeptide which is at least 10 times greater, preferably 100 to 1000 times greater, than the concentration of Otx2.
  • said polypeptide may be used at a concentration of from 1 to 10 ⁇ M, advantageously from 10 to 100 ⁇ M, and particularly advantageously from 100 to 1000 ⁇ M.
  • said targeting polypeptide will preferably be administered locally, for example by means of an osmotic minipump implanted in the brain.
  • chimeric polypeptide to be administered in vivo in order to obtain the desired concentration on contact with the target cells can be readily determined and adjusted by those skilled in the art depending, in particular, on the administration methods envisaged.
  • Cell targeting polypeptides in accordance with the invention can also be used to screen for other molecules capable of binding specifically to Otx2 target cells.
  • a subject of the present invention is a method of screening for molecules capable of binding specifically to Otx2 target cells, at the same binding sites as Otx2, characterized in that it comprises:
  • said method is carried out in the presence both of Otx2 target cells and of cells which do not bind Otx2, for example on a section of retina.
  • chimeric proteins comprising an alkaline phosphatase domain coupled to the whole Otx2 protein or to various fragments of this protein were constructed.
  • the sequences encoding the human Otx2 protein or encoding the fragments tested were cloned into the vector pAPtag-5 (GenHunter), in frame with the sequence encoding alkaline phosphatase.
  • the various constructions carried out are represented diagrammatically in FIG. 1 .
  • signal seq. signal peptide of alkaline phosphatase
  • Alkaline Phosphatase alkaline phosphatase
  • Nt N-terminal region of Otx2 (amino acids 1-37);
  • Hd homeodomain of Otx2 (amino acids 38-97);
  • Ct C-terminal region of Otx2 (amino acids 98-289);
  • 6 ⁇ His polyhistidine tag.
  • HEK 293 cells cultured in culture dishes 10 cm in diameter were transfected with 10 ⁇ g of each of the vectors constructed, purified beforehand, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
  • the transfected cells were incubated for 48 hours in DMEM/F12, supplemented with 10% of fetal bovine serum (FCS).
  • FCS fetal bovine serum
  • the supernatant was collected, centrifuged at 100 ⁇ g for 5 minutes and stored at ⁇ 80° C. This supernatant is used, without purification, for testing the binding of the chimeric proteins on sections of retina.
  • cryostat sections (20 ⁇ m) of frozen fresh retinas are fixed for 8 minutes in 100% cooled methanol, and then washed 3 times 10 in phosphate buffer (PBS) with 4 mM of MgCl 2 .
  • PBS phosphate buffer
  • the sections are incubated in PBS buffer, 4 mM MgCl 2 , plus 10% FCS for 1 hour at room temperature (RT).
  • the supernatants containing the fusion proteins tested are diluted to 1/20 in PBS and incubated for 2 hours at room temperature.
  • the sections are then washed 5 times in PBS, 4 mM MgCl 2 , and the bound ligands are then fixed for 2 minutes (60% acetone, 4% PFA, 20 mM Hepes, pH 7).
  • the sections are heated at 65° C. for 2 hours in PBS in order to inactivate the endogenous phosphatases.
  • the sections are then washed twice in PBS before visualization of the alkaline phosphatase activity (preincubation of the sections for 5 minutes in 100 mM Tris, pH 9.5, 100 mM NaCl, 5 mM MgCl 2 , followed by the addition of NBT/BCIP (Promega)).
  • FIG. 2 The results obtained with the AP-Nt-Otx2 and AP-Hd-Otx2 fusion proteins are illustrated by FIG. 2 .
  • CP Cells of the retinal pigment epithelium
  • Cb bipolar cells
  • RGC ganglion cells
  • Biotinylated peptides corresponding to various fragments of the N-terminal domain+homeodomain portion of Otx2 were synthesized and the binding thereof to sections of retina, prepared as described above, was tested.
  • the incubation of the peptides with the sections of retina was carried out under the same conditions as those described above, and then the sections bearing the bound peptides were incubated with alkaline phosphatase-labeled streptavidin, and the alkaline phosphatase activity was detected as described above.
  • RK-Otx2 One of the peptides tested (RK-Otx2), corresponding to the sequence RKQRRERTTFTRAQL (SEQ ID NO: 2), has the same binding specificity as the AP-Otx2 and AP-Nt-Otx2 fusion proteins.
  • CP Cells of the retinal pigment epithelium
  • Cb bipolar cells
  • RGC ganglion cells
  • RK-Otx2 peptide binds, like AP-Nt-Otx2, to the ganglion and bipolar cells.
  • no binding is observed with the AA-Otx2 peptide.
  • Another peptide (RA-Otx2), in which the RK dipeptide is replaced with RA, binds only very weakly to the ganglion and bipolar cells (results not shown).
  • CP Cells of the retinal pigment epithelium
  • Cb bipolar cells
  • RGC ganglion cells
  • Otx2 protects retinal ganglion neurons against the toxic effects of N-methyl-D-aspartate (NMDA).
  • a chimeric polypeptide was constructed genetically and produced by bacterial synthesis, by fusing the N-terminal domain of Otx2 and its homeodomain (amino acids 1-97 of Otx2), with the VP16 trans-activator domain of the herpes virus (MLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG, SEQ ID NO: 4).
  • C57 B16 mice received, in the right eye, 1 ⁇ l of injection buffer (PBS or 9 ⁇ NaCl) containing either 1 mM of NMDA, or 1 mM of NMDA supplemented with 30 ng of the chimeric polypeptide, and in the left eye, the same volume of injection buffer, without additive.
  • injection buffer PBS or 9 ⁇ NaCl
  • the survival of the ganglion neurons was determined by measuring the level of expression of Brain 3A (Brn3A), a transcription factor which, in the retina, is specifically expressed in the ganglion neurons (Xiang et al., J. Neurosci., 15, 4762-4785, 1995).
  • Brn3A Brain 3A
  • the animals are sacrificed, the retinas are removed, and the mRNA is extracted therefrom.
  • the level of expression of Brn3A mRNA was determined by quantitative RT-PCR using the hypoxanthine phosphoribosyltransferase (HPRT) gene as reference gene, and the ratio between the expression of the Brn3A mRNA in the right eye and in the left eye was calculated.
  • HPRT hypoxanthine phosphoribosyltransferase
  • FIG. 5 The results are illustrated by FIG. 5 .
  • the additives used are indicated along the x-axis; the ratio between the amounts of Brn3A mRNA (standardized relative to the HPRT mRNA) in the right eye and in the left eye is indicated along the y-axis.
  • AP-Nt-Otx2 being a variant of AP-Nt-Otx2 in which the RK amino acid doublet has been replaced with the AA doublet
  • AP-Nt-Otx2 alone (culture supernatant diluted to 1/20) or in the presence of a whole Otx2 (1 ⁇ g/ml) or of the RK-Otx2 or AA-Otx2 peptides (2 ⁇ g/ml)
  • A-D scale bar 500 ⁇ m;
  • A AP-Nt-Otx2;
  • B AP (alkaline phosphatase alone);
  • C AP-Nt-Otx2 in the presence of a whole Otx2;
  • D AP-Hd-Otx2;
  • E-H scale bar 100 ⁇ m;
  • E AP-Nt-Otx2;
  • F AP-HdAA-Otx2;
  • G AP-Nt-Otx2 in the presence of RK-Otx2;
  • H AP-Nt-Otx2 in the presence of AA-Otx2.
  • Glycosaminoglycans GAGs
  • chondroitin sulfate proteoglycans are an essential constituent of the extracellular matrix (perineuronal net) which surrounds the parvalbumin neurons of the visual cortex.
  • the putting into place of this perineuronal net coincides with the end of the critical period of plasticity; it constitutes a major factor in the loss of cortical plasticity that occurs at the end of this critical period, and it has been shown that the destruction of this perineuronal net by treating with chondroitinase-ABC makes it possible to restore this plasticity (Pizzorusso et al., Science, 298, 1248-51, 2002).
  • AP-Otx2 In order to determine whether the perineuronal net associated with parvalbumin neurons was involved in the binding between AP-Nt-Otx2 and its target cells, the binding of AP-Otx2 was tested on cryostat sections of adult mouse brains fixed with methanol, and then incubated for 24 hours in the presence of chondroitinase ABC (2 U/ml) in a buffer containing 50 mM Tris [pH 8.0], 40 mM of sodium acetate, 0.1% BSA and protease inhibitors.
  • the RK-Otx2 peptide can block the binding of Otx2 to its target cells, in vitro. It was tested whether this effect also occurred in vivo.
  • osmotic minipumps Alzet 1003D, Alza
  • mice are perfused with 4% PFA, and brain sections (25 um) are cut in order to study the localization of Otx2 and that of the RK-Otx2 peptide.
  • Otx2 is visualized using a rat anti-Otx2 monoclonal antibody diluted to 1/200 (Sugiyama et al., Cell, 134, 508-20, 2008), followed by a donkey anti-rat antibody labeled with Alexa 488 (Molecular Probes), diluted to 1/2000.
  • Alexa 488 Molecular Probes
  • the cells expressing Otx2 were counted over a surface area of
  • FIG. 8 The results are illustrated by FIG. 8 .
  • A, B Detection of the RK-Otx2 peptide; A: control hemisphere; B: infused hemisphere.
  • E Quantification of the cells expressing Otx2; black bars: control hemisphere; gray bars: infused hemisphere.
  • F, G Detection of the AA-Otx2 peptide; A: control hemisphere; B: infused hemisphere.
  • H I: Detection of Otx2; H: control hemisphere; I: infused hemisphere.
  • the RK-Otx2 peptide or the AA-Otx2 peptide was infused in adult mice (therefore after closing of the critical period of plasticity), as described in example 4 above. Chondroitinase-ABC, or the injection buffer (deionized water+0.1% BSA), were injected (0.4 ⁇ l for each injection) at 3 sites surrounding the visual cortex (AP lambda, LM 1.5 mm; AP lambda, 4.0 mm; AP+1.5 mm; LM 2.5 mm) at two different depths (300 and 500 ⁇ m).
  • mice After infusion of the peptides or injection of chondroitinase ABC, the mice are subjected to monocular deprivation for 4 days, and the responses to visual stimuli are then measured by single-unit extracellular electrophysiology.
  • the electrophysiology recordings are carried out under nembutal/chlorprothixene anesthesia using standard techniques (Gordon & Stryker, J Neurosci, 16, 3274-86, 1996; Mataga et al., Neuron, 44, 1031-41, 2004). 5 to 7 single-unit recordings were carried out for each mouse, on both sides of the medial-lateral axis of the primary visual cortex, in order to cover the monocular zone and the binocular zone, and to avoid sampling biases.
  • CBI contralateral bias index
  • This weighted mean of the bias in favor of one or other eye can range from 0, for complete ipsilateral dominance, to 1, for complete contralateral dominance.
  • mice brain frontal sections were prepared, as described in example 4 above, in order to determine the influence of the RK-Otx2 peptide on parvalbumin neurons.
  • the sections were labeled either with WFA, as described in example 3, or using a mouse anti-parvalbumin monoclonal antibody ( 1/500, Sigma-Aldrich), which was visualized using a donkey anti-mouse antibody labeled with Cy3.
  • the labeled cells were quantified as described in example 4.
  • A Contralateral bias index after infusion of the RK-Otx2 peptide (RK) or of the AA-Otx2 peptide (AA), or after injection of chondroitinase ABC (chABC) or of injection buffer alone (Veh).
  • the infusion of the RK-Otx2 peptide decreases the expression of the sites for the binding of WFA, and also that for parvalbumin.
  • the number of parvalbumin-positive cells decreases by 56.2%, and that of cells surrounded by sites for the binding of WFA decreases by 51.3%.

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KR102098006B1 (ko) * 2018-02-23 2020-04-07 삼성전자주식회사 외부 객체를 감지하여 무선 전송 전력을 제어하는 방법 및 이를 구현한 전자 장치
WO2022129976A1 (fr) 2020-12-17 2022-06-23 Sorbonne Universite Polypeptides se liant sélectivement à des glycosaminoglycanes de type héparine ou sulfate d'héparane et polypeptides de pénétration cellulaire comprenant ceux-ci

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US10722554B2 (en) * 2008-01-09 2020-07-28 Centre National De La Recherche Scientifique Methods of reducing excitotoxicity-induced retinal ganglionic neuron degeneration by an orthodenticle HOMOLOG2 (OTX2) homeoprotein

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CA2750005C (fr) 2018-02-27
WO2010081975A1 (fr) 2010-07-22
EP2387579A1 (fr) 2011-11-23
CA2750005A1 (fr) 2010-07-22
JP5756024B2 (ja) 2015-07-29
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US10842852B2 (en) 2020-11-24
FR2941230A1 (fr) 2010-07-23
ES2471458T3 (es) 2014-06-26

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