Method and pharmaceutical composition for use in the treatment of neurodegenerative disorders

Abstract

The invention relates to compounds which activate the BASIGIN signalling pathway, preferably agonists of BASIGIN, for the treatment of neurodegenerative disorders.

Claims

1. A method for screening BASIGIN pathway activating compounds for the treatment of a retinal neurodegenerative disorder comprising the steps of: a) providing a plurality of neurons expressing BASIGIN on their surface, wherein said neurons are cone photoreceptors, wherein said BASIGIN contains three immunoglobulin-like domains in its extracellular portion; b) incubating said neurons with a candidate compound; c1) determining whether said candidate compound binds to BASIGIN and c2) determining whether said candidate compound activates BASIGIN by: determining neuron viability, wherein an increased viability of neuron in a culture medium comprising the candidate compound compared to a control without candidate compound being indicative of an activation of BASIGIN; and/or analyzing a downstream molecular signaling pathway by measuring an intracellular ATP content, wherein an increased intracellular ATP content compared to a control without candidate compound being indicative of an activation of BASIGIN; thereby determining whether said candidate compound activates BASIGIN; and d) selecting the candidate compound that binds to and activates said BASIGIN.

2. The method of claim 1 wherein the retinal neurodegenerative disorder is retinitis pigmentosa.

Description

FIGURES

(1) FIG. 1: Specific binding for the cone-enriched culture: Iodinated human RdCVF peptide was incubated with a. cone-enriched cultures from chicken embryo, b. retinal pigmented epithelial cells from pig, c. Cos-1 cells. The dash line in a shows the specific binding after competition with the mouse unlabelled peptide

(2) FIG. 2: Purification of the RdCVF receptor by far-western blotting assay: Fractions from chicken retina and embryonic fibroblasts (CEF). S, solubles. M, membrane fractions. T total. Lane 2-4 coomassie staining. Lanes 5-14 farwestern blotting with GST-RdCVFL, GST-RdCVF and GST as indicated

(3) FIG. 3: Migration of the receptor BASIGIN on SDS-PAGE: Analysis by western blotting of retinal extracts (taken from published work)

EXAMPLES

Example 1

Specific Binding for the Cone-Enriched Culture

(4) Material & Methods:

(5) Human RdCVF (hRdCVF) and mouse RdCVF (mRdCVF) were synthesised at GeneProt and refolded (>90% purity). The chloramine T method was used to label hRdCVF with .sup.125I with a specific activity of 2130 Ci/mmol. Chick retina cells were isolated and cultured as previously described (Adler and Hatlee, 1989; Fintz et al, 2003) with minor modifications. The cells were plated on poly-L-lysine coated 24-well plates at 3×10.sup.5 cells/cm.sup.2 (6×10.sup.5/well) and cultured for 24 h in serum-containing medium. After 24 hours, the cells were rinsed three times then, 50 μl binding buffer (control) or 50 μl binding buffer containing unlabelled mRdCVF (to determine non-specific binding) was added to each well of the culture. After incubation for 30 min at room temperature, 50 μl binding buffer containing [.sup.125I]-hRdCVF was added to each well. After incubation for 90 min at room temperature, the plates were incubated at 4° C. for 45 min. The cells were rinsed three times and solubilised with 1% SDS. The radioactivity of the extracts was counted using a gamma counter. The specific binding was measured by competition assay with excess unlabelled recombinant mouse RdCVF. The level of radioactivity was partially reduced by 300 nM unlabelled mRdCVF suggesting the presence of specific sites, despite saturation not being reached in the range of [.sup.125I]-hRdCVF concentrations (0.05-0.5 nM, FIG. 2a). In three independent experiments, 300 nM mRdCVF inhibited 26% (2.4 fmol/well), 32% (1.2 fmol), and 31% (0.76 fmol) of the measured radioactivity at 0.5 nM [.sup.125I]-hRdCVF.

(6) Results:

(7) In the absence of cells, we observed no inhibitory effect of mRdCVF suggesting that specific binding occurred on cells. We calculated a specific binding with a Kd of about 150 pM and a Bmax of 200 sites/cell. Cell differentiation seems to be involved because we could not detect RdCVF binding sites in fresh cell suspensions. Although the lack of binding of undifferentiated cells may be reflecting the specificity of the putative RdCVF receptor expression by cones, we address this directly by comparing the binding on chicken cones to that on primary retinal pigmented epithelial (RPE) cells from pigs. As RPE cells do not respond to RdCVF trophic activity, we expect them to show no specific RdCVF binding. The radioiodination of the synthetic RdCVF peptide was performed with an alternative protocol to that previously used (iodo-beads instead of lactoperoxydase). We have compared the binding of [.sup.125I]-hRdCVF and the inhibitory effect of mouse RdCVF to cone-enriched cells from chicken retina, retinal pigmented epithelium (RPE) from pig and COS-1 cells. The non specific binding is linear for the three types of cells examined. Specific binding was observed only for the cone-enriched culture (FIG. 1). A comparison between [.sup.125I]-hRdCVF binding and ATP content in four independent experiments show that there is a linear relationship between total binding and ATP. The IC50 was estimated to 35 nM (5.7 to 210 nM, 95% confidence limits). A rough estimation of the number of saturable binding sites, obtained by extrapolation of the binding parameters, gives 54,000 per cell (5,300 to 460,000, 95% confidence limits). This confirms that a specific binding activity for RdCVF is expressed by cones.

Example 2

Purification of the RdCVF Receptor by Far-Western Blotting Assay

(8) Material & Methods:

(9) To purify the RdCVF receptor from the membrane fractions of the chicken retina, we have developed a far-western blotting assay (Léveillard et al., 1996). The protein fractions loaded onto an SDS gels are transferred onto nitrocellulose membranes. The denatured proteins on the membrane are then partially re-natured by serial incubation in buffer containing decreasing amounts of guanidine, the denaturing agent. The protein, used here as a probe, is produced in E. coli as a GST fusion in the vector pGEX2TK. We have previously shown that RdCVF protein produced using this vector system has a trophic activity toward chicken cones (Leveillard et al., 2004). This probe was applied to proteins on the membrane (FIG. 2).

(10) Results:

(11) We have looked for RdCVF-interacting proteins in the membrane and soluble fractions of chicken embryonic retina and test the tissue restricted expression using membrane fraction from chicken embryonic fibroblasts (CEF). We have used both the long and the short RdCVF proteins as probes as well as GST. When the protein GST was used as negative control, two non specific bands could be detected (*=37, 28 kDa in lane 14). Interestingly, several specific interacting proteins were found the membrane fractions of chicken retina (+˜100, 75 and 45 kDa in lane 6 for GST-RdCVFL of chicken retina). The band at 50 kDa is likely corresponding to a soluble protein contaminating the membrane fraction since it is enriched in lane 5. The band at about 60 kDa, detected in lane 6, has a broader range of expression since it is also detected in the membrane fraction of chicken embryonic fibroblast (triangle in lane 8). Using the same extracts the short isoform detects a specific band smaller than 45 kDa in the membrane fraction of chicken retina (+in lane 10). The coomassie staining of these fractions shows that there is no prominent band at 45 kDa (lanes 2-4). Taken together these results indicate the existence of a candidate RdCVF interacting protein in the membrane fraction of the chicken retina migrating at 45 kDa. Additionally, while non quantitative, our assay shows that this interaction is of higher affinity with the short (lane 10) than that long (lane 6) isoform of RdCVF. We have shown that the trophic effect is mediated by the short and not the long isoform (Léveillard et al., 2004 and data not shown) reinforcing our interest in this 45 kDa protein.

(12) We have performed a proteomic analysis of the band at 45 kDa from membrane fraction of chicken retina (lane 3). Because of the limited purification (membrane fractionation and SDS page), each band contains many proteins. A total of 144 proteins were identified using MS/MS for at least one matching peptide. The analysis of 111 proteins with sequence covered by at least 5%, shows that 58 proteins were identified as protein from the Gallus gallus genome and were considered for further analysis. Within that list, the presence of arrestin, vimentin and cone-type transducin alpha subunit signs the presence of retinal proteins within this fraction but also indicates that the protocol of purification does not allow removing all soluble proteins. A short list of 33 candidate proteins was established by removing unlikely candidates as the three retinal protein cited and other abundant and most likely contaminating proteins as enolase, creatine kinase, beta actin, mitochondrial ATPase, translation initiation factor, aldolase, Glucose 3P deshydrogenase, glucose phosphate isomerase, pyruvate kinase, heterogenous nuclear ribonucleoproteins as described in Casuvoglu et al. (2003). The abundance of proteins linked to the inner membrane of the cells as G-protein probably reflects the abundance of proteins associated with cell surface receptors in our preparation.

(13) Among the list of 33 candidate proteins, 3 proteins containing a transmembrane domain (TM), a criterion that we have retained for the selection of a cell surface receptor. One of those was the cell surface glycoprotein HT7 precursor also known as BASIGIN (gi|69992).

Example 3

Migration of the Receptor BASIGIN on SDS-PAGE

(14) The cell surface glycoprotein BASIGIN was identified was originally identified as a blood brain barrier protein identical to the 5A11 antigen that mediates the cell-cell recognition in the avian retina (Fadool and Linser, 1993), a monoclonal antibody generated by immunizing mice with live cells dissociated from isolated day 7 embryonic chick retina (Linser and Perkins, 1987). Those cells are very similar to the day 6 embryonic chick retina used as cone-enriched culture system (Fintz et al., 2003; Léveillard et al., 2004). The migration of this glycoprotein on SDSPAGE is very similar to the RdCVF interacting protein as seen in a figure taken from the work of Fadool and Linser (FIG. 3). Interestingly, in that report the authors show that BASIGIN antibody (clone 5A11) inhibits retina cell reaggregation in vitro supporting the role of BASIGIN in cell-cell interactions. We have identified two clones encoding BASIGIN from an expression library constructed with RNA prepared from our cone-enriched cultures. The presence of these two clones out of a total of 1000 EST sequenced within the frame of an ongoing collaboration with the Génoscope (http://www.genoscope.cns.fr/spig/Mus-musculusdeqeneration-of.html) indicates that most likely BASIGIN is expressed by cones.

Example 4

Neuron Rescue Activity of BASIGIN Agonists

(15) The BASIGIN agonists described in the present invention are tested for their ability to rescue neurons according to the following protocols:

(16) 1) Cone Rescue Activity

(17) The primary culture is a cone-enriched primary cell culture system from chicken embryo (60-80% of cones) as described in Fintz et al. (Invest. Ophtamol. Vis. Sci, vol 44(2): 818-825, 2003.)

(18) After 7 days of incubation with or without test compound, a period over which these post-mitotic cells degenerate, the viability of the cells in the culture is scored using the Live/Dead assay (Molecular Probes) and a cell counting platform as previously described (Leveillard et al., 2004).

(19) 2) Olfactory Sensitive Neuron Rescue Activity

(20) Adult mice are killed by decapitation. The posterior part of the nasal septum is dissected free of the nasal cavity and immediately placed in ice-cold DMEM containing 50 μg/ml gentamicin (Eurobio; Gibco) and 10 (v/v) fetal calf serum (eurobio). The cartilage of the septum is removed and the olfactory mucosa is incubated for 30 min at 37° C. in a 2.4 U/MI dispase II solution (Roche. The olfactory epithelium is carefully separated from the underlying lamina propria under the dissection microscope and gently triturated about 20 times to separate the cells. The resulting cell suspension is transferred to a 50 ml conical tube and the dispase is inactivated by adding 40 ml of HBSS without calcium and magnesium. The cell suspension is centrifuged at 700 rpm for 5 min, and the pellet containing the cells is resuspended in a medium composed of DMEM containing insulin (10 μg/ml, Sigma), transferin (10 μg/ml, Sigma), selenium (10 μg/ml, Sigma) calf foetal serum (5%), ascorbic acid (200 μM). Cells are plated on 12 mm sterile glass coverslips coated with 5 μg/cm.sup.2 human collagen IV (Sigma); thus providing a primary culture of Olfactory Sensitive Neurons (OSN).

(21) After 4 days of culture with or without test compound, cells are fixed and labelled with tubulin III, and counted.

(22) 3) Purkinje Cell Rescue Activity

(23) After decapitation of mouse at postnatal day 1-3, brains are dissected out into cold Grey's balanced salt solution containing 5 mg/ml glucose, and the meninges are removed. Cerebellar parasagittal slices (35° or 250 μm thick) are cut on a Mcllwain tissue chopper and transferred onto membrane of 30 mm Millipore culture inserts with 0.4 μm pore size (Millicel; Millipore, Bedford, Mass.). Slices are maintained in culture in 6-well plates containing 3 ml of medium at 35° in an atmosphere of humidified 5% CO.sub.2. The medium is composed of 50% basal medium with Earle's salts (Invitrogen), 25% HBSS (Invitrogen), 25% horse serum (Invitrogen), L-glutamine (1 mM) and 5 mg/ml glucose (Stoppini et al., J Neurosci Methods, vol 37(2), p 173-82, 1991).

(24) After 4 days of culture with or without test compound, cells are fixed and labelled with tubulin III, and counted.

(25) 4) Cortical Neuron Rescue Activity

(26) Serum-free preparation of mouse cortical primary cultures is performed with mouse at postnatal day 1. After removal of the meninges, entire cortices are mechanically dissociated un a phosphate buffer saline glucose solution without added divalent cations (100 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 7.9 mM Na2HPO4, 33 mM glucose, 100 U/ml penicillin and 100 μg/ml streptomycin) and resuspended in Neurobasal medium (Invitrogen) containing 2% B27 supplement (Gibco), 0.5 mM glutamine; and 25 μM glutamate. Cells are then cultured onto poly-ornithine coated coverslips to produce cultures highly enriched in cortical neurons.

(27) After 4 days of culture with or without test compound, cells are fixed and labelled with tubulin III, and counted.

REFERENCES

(28) Arner and Holmgren, Eur. J. Biochem., vol. 267(20), p: 6102-6109, 2000. Carter-Dawson et al., Invest. Ophthalmol. Vis. Sci., vol. 17(6), p: 489-498, 1978. Holmgren, J. Biol. Chem., vol. 254(18), p: 9113-9119, 1979. Holmgren, J. Biol. Chem., vol. 254(19), p: 9627-9632, 1979. Holmgren, Annu. Rev. Biochem., vol. 54, p: 237-271, 1985. Holmgren, J. Biol. Chem., vol. 264(24), p: 13963-13966, 1989; Jeffery, Trends Biochem. Sci., vol. 24(1):8-11, 1999. Jeffery, Trends Genet., vol. 19(8):415-417, 2003. Leveillard et al., Nat. Genet. vol. 36(7), p: 755-759, 2004). Liu et al., Blood, vol. 105(4):1606-1613, 2005. Mohand-Said et al., Proc. Natl. Acad. Sci. U.S.A, vol. 95(14), p: 8357-8362, 1998). Pekkari et al., J. Biol. Chem., vol. 275(48), p: 37474-37480, 2000. Pekkari et al., FEBS Lett., vol. 539(1-3):143-148, 2003. Pekkari et al., Blood, vol. 105(4), :1598-1605, 2005. Portera-Cailliau et al., Proc. Natl. Acad. Sci. U.S.A, vol. 91(3), p: 974-978, 1994. Powis and Montfort, Annu. Rev. Pharmacol. Toxicol., vol. 41, p: 261-295, 2001.