AN UBIQUITIN LIGASE INHIBITOR FOR USE FOR PREVENTING AND/OR TREATING A DISEASE LINKED WITH CEREBRAL HYPOPERFUSION

Abstract

The present invention concerns an ubiquitin ligase inhibitor for use for preventing and/or treating a disease linked with cerebral hypoperfusion, and an in vitro screening method for the identification of a candidate compound suitable for preventing and/or treating a disease linked with cerebral hypoperfusion.

Claims

1. A method for preventing and/or treating a disease linked with cerebral hypoperfusion.

2. The method according to claim 1, wherein the disease is selected from vascular dementia, Parkinson's disease, Multiple sclerosis or Alzheimer's disease.

3. The method according to claim 1, wherein said inhibitor is an inhibitor of Pdzrn3 expression.

4. The method according to claim 3, wherein said inhibitor is chosen from small interfering (siRNA), small hairpin RNA (shRNA), micro RNA (miRNA), antisense oligonucleotides and aptamers.

5. The method according to claim 1, wherein said inhibitor inhibits PDZRN3 biological activity or inhibits PDZRN3 interaction with its targets.

6. The method according to claim 5, wherein said inhibitor is chosen from chemical molecules, peptides, proteins, aptamers, antibodies and antibody fragments.

7. The method according to claim 1, wherein said inhibitor reduces brain inflammation.

8. The method according to claim 1, wherein said inhibitor is fused or linked to a tag allowing the targeting of cerebral endothelial cells.

9. The method according to claim 1, wherein said inhibitor is formulated in a pharmaceutical composition.

10. The method according to claim 1, wherein said inhibitor is administered by oral route, intranasal route, intraocular route, parenteral route, or by intramuscular, subcutaneous, intravenous, intraperitoneal or local injections.

11-12. (canceled)

13. An in vitro screening method for the identification of a candidate compound suitable for preventing and/or treating disease linked with cerebral hypoperfusion, said method comprising: a. culturing endothelial cells, in the presence and in the absence of a candidate compound; b. measuring the level of expression of Pdznr3 or the level of PDZRN3 biological activity in endothelial cells cultured in the presence and in the absence of the candidate compound; c. comparing the level of expression of Pdznr3 in endothelial cells in the presence the candidate compound, with the level of expression of Pdznr3 endothelial cells in the absence of the candidate compound; or comparing the level of PDZRN3 biological activity in endothelial cells in the presence the candidate compound, with the level of PDZRN3 biological activity in endothelial cells in the absence of the candidate compound; and d. identifying the candidate compound as suitable for preventing and/or treating disease linked with cerebral hypoperfusion if the level of expression of Pdznr3 in endothelial cells in the presence the candidate compound is decreased compared with the level of expression of Pdznr3 in endothelial cells in the absence of the candidate; or identifying the candidate compound as suitable for preventing and/or treating disease linked with cerebral hypoperfusion if the level of PDZRN3 biological activity in endothelial cells in the presence the candidate compound is decreased compared with the level PDZRN3 biological activity in endothelial cells in the absence of the candidate.

14. A pharmaceutical composition comprising a PDZRN3 inhibitor and a pharmaceutically acceptable carrier.

Description

FIGURES

[0067] FIG. 1.

[0068] Experimental scheme for tamoxifen administration to postnatal Pdzrn3 iECKO and IECOE mice; timeline for evaluation of spatial recognition memory in the Y-maze paradigm before and after implantation of ameroid constrictors (BCAS) is shown.

[0069] FIG. 2.

[0070] Spatial recognition memory performance in the Y-maze exploration test;

[0071] left side: deletion of Pdzrn3 (iECKO) protects against chronic hypoperfusion-induced loss of spatial novelty preference in Y-maze evaluated 10 days after BCAS.

[0072] right side: Ectopic expression of Pdzrn3 (iECOE) worsened recognition memory performance evaluated 7 days after BCAS.

[0073] Results are presented as a recognition index calculated as follows: (time in novel arm−time in familiar arms)/(time in novel arm+time in familiar arms). Student's t-test was performed. Each group contained 9-16 mice and each mouse is indicated as a dot in the graphs.

[0074] FIG. 3.

[0075] NeuN+ cells were quantified as a percentage of the area occupied in the hippocampal CA1 area before (sham) and after BCAS from iECKO vs their respective littermate mice (left side) and from iECOE vs their respective control mice (right side). Data shown in the graphs are obtained in sham condition, from 3 mice per group; in BCAS condition, from n=13 IECKO vs n=15 littermate mice and from n=6 ICOE vs n=5 control mice.

[0076] FIG. 4.

[0077] GFAP were quantified in hippocampal area after BCAS in iECKO (left side) and iECOE (right side) mice versus their respective littermates. The size of each group is the same as indicated in FIG. 3.

[0078] FIG. 5.

[0079] Fibrinogen release was quantified as a percentage of the area occupied in hippocampal region from Pdzrn3 iECKO (n=4) versus littermates (n=7) mice at 21 days after BCAS. Unpaired t-test was performed. *P 0.05. Data are presented as mean±s.e.m (Student's t-test).

[0080] FIG. 6.

[0081] Fibrinogen was quantified as a percentage of the area occupied either in cortical region (left side) or in hippocampal region (right side) before (sham) and after BCAS.

[0082] Quantification was done in brains from non operated sham control (n=3) vs iECOE (n=3) mice and in brains recovered after 14 days of BCAS from control (n=7) vs iECOE (n=6) mice. Data for two brain slices per mouse were recorded and averaged to produce a single value for each mouse. One-way ANOVA was performed. Data are presented as mean±s.e.m.

[0083] FIG. 7.

[0084] IgG was quantified as a percentage of the area occupied either in cortical region (left side) or in hippocampal region (right side) before (sham) and after BCAS.

[0085] Quantification was done in brains from non operated sham control (n=3) vs iECOE (n=3) mice and in brains recovered after 14 days of BCAS from control (n=7) vs iECOE (n=6) mice. Data for two brain slices per mouse were recorded and averaged to produce a single value for each mouse. One-way ANOVA was performed. Data are presented as mean±s.e.m.

[0086] FIG. 8.

[0087] Y-maze exploration test: deletion of Pdzrn3 (iECKO) protects against AD-induced loss of spatial novelty preference in Y-maze evaluated in APP/PS1; IECKO vs APP/PS1 groups at 6, 8 and 10 months of age. Each point represents a different mouse. Data are presented as mean±s.e.m. Recognition index calculated as described in FIG. 2. Two-way ANOVA was performed.

[0088] FIG. 9.

[0089] Surface of amyloid plaque staining was quantified in entire cortex with the anti-amyloid monoclonal 6E10 antibody. One-way ANOVA was performed. Data are presented as mean±s.e.m.

[0090] FIG. 10.

[0091] Surface of amyloid plaque staining was quantified in hippocampal region with the anti-amyloid monoclonal 6E10 antibody. One-way ANOVA was performed. Data are presented as mean±s.e.m.

[0092] FIG. 11.

[0093] GFAP positive staining was quantified as a percentage of the area occupied in cortical region in APP/PS1; iECKO vs their age-matched littermates APP/PS1 at 6 and 12 months. Each point represents a different mouse. Data are presented as mean±s.e.m. Two-way ANOVA was performed.

[0094] FIG. 12.

[0095] NeuN positive staining was quantified as a percentage of the area occupied in cortical region in APP/PS1; iECKO vs their age-matched littermates APP/PS1 at 6 and 12 months. Each point represents a different mouse. Data are presented as mean±s.e.m. Two-way ANOVA was performed.

[0096] FIG. 13.

[0097] IgG extravasion was quantified as a percentage of the area occupied in cortical region in APP/PS1; iECKO vs their age-matched littermates APP/PS1 at 6 and 12 months. Each point represents a different mouse. Data are presented as mean±s.e.m. Two-way ANOVA was performed.

[0098] FIG. 14.

[0099] Transcript levels in brain capillaries isolated from either non-operated ND-iECKO (n=5) vs ND-littermates (n=5) and APP/PS1; iECKO (n=4) vs APP/PS1 (n=5) mice. Data are normalized to that of PO and presented as the relative fold to sham. One-way ANOVA was performed.

[0100] *P≤0.05; **P≤0.01; ****P≤0.0001.

EXAMPLES

[0101] Brain endothelial loss of Pdzrn3 protects against induced BBB dysfunction and cognitive impairment whereas endothelial Pdzrn3 ectopic expression enhances brain damage in mice with chronic cerebral hypoperfusion.

[0102] Given that Pdzrn3 function is linked with reduced endothelial integrity, the inventors studied whether the cerebral phenotypes caused by chronic HP can be rescued by repression of or worsened by ectopic Pdzrn3 expression in central nervous system (CNS) endothelial cells (EC).

[0103] To recapitulate chronic HP insult, an experimental murine bilateral carotid artery stenosis (BCAS) model system was developed with ameroid constrictors implanted on both common carotid arteries (FIG. 1). Cortical surface Cerebral blood flow (CBF) was then recorded and a continued CBF decrease was observed at 21 days after surgery. Mice bearing a Pdzrn3 flox allele (Pdzrn3.sup.f/f) were crossed with an EC-specific Pdgfb-CreER transgene to generate Pdzrn3.sup.f/f; Pdgfb-iCreER (iECKO: inducible Endothelial Cells Knock Out) and Pdzrn3.sup.f/f littermates (control). For this experiment recombination was induced with CreER and 4-hydroxytamoxifen (4HT) treatment 5 weeks after birth and animals were analyzed at 5 months (FIG. 1). Recombination efficiency in the brain was determined by analyzing Pdgfb-CreERT2; mTmG reporter mice showing that Pdgfb drives efficiently endothelial recombination in brain vessels. To avoid any effect of endothelial Pdzrn3 deletion on microvessel organization in CNS, the vasculature in brain volumes of iECKO mice vs control littermates was studied by light sheet microscopy.

[0104] Both Pdzrn3 iECKO and control littermate groups were then subjected to behavioral testing. To verify that the results from the cognitive tests were not confounded by differences in sensorimotor abilities between the animals, sensorimotor testing was first conducted. No statistical differences were observed between the groups in locomotor activity in terms of distance traveled, maximum speed, mean velocity. The two groups were subsequently tested, for spatial recognition memory in the Y-maze. Pdzrn3.sup.f/f littermate group spent significantly less time in the novel arm compared to the iECKO group after BCAS at 10 days. Thus, Pdzrn3 deletion rescued the memory disturbance (FIG. 2, left side).

[0105] The effect of the endothelial Pdzrn3 deletion was studied on chronic HP-induced brain lesions and inflammation. Compared to controls, iECKO mice exhibited a significant decrease in the number of brain lesions (microinfarctions) as well as reduced neuronal loss in the CA1 hippocampal region known to play a crucial role in spatial recognition memory (FIG. 3, left side). To evaluate the effect of the Pdzrn3 deletion on brain inflammation, hippocampal GFAP expression was quantified, it is a marker of reactive astrocytes. Chronic HP induced an activation of astrocytes; loss of Pdzrn3 decreases GFAP positive labelling in the hippocampus (FIG. 4, left side).

[0106] Capillary leakage is a hallmark of altered BBB and found in clinic of AD or vascular dementia. To analyze BBB disruption under HP, hippocampal sections were triple stained for the endothelial and astrocyte markers, respectively Podocalyxin and GFAP, and with Fibrinogen to assess blood extravasation across leaky BBB. A rupture of both endothelial and astrocyte layers with extravasation of Fibrinogen was found in littermate while Pdzrn3-deficient mice showed reduced capillary rupture mostly in cortex and hippocampal regions. Furthermore, a higher accumulation of fibrinogen was found in mouse brain from littermates compared to iECKO mice under chronic HP (FIG. 5).

[0107] Maintenance of the BBB might requires functional interactions between endothelial cells, perivascular cell and astrocytes. Whole gliovascular unit transcripts were then analyzed by qRT-PCR in preparation of intact brain vessel fragments from the mouse brain parenchyma. Under 14 days of chronic HP, an increase of plasmalemma vesicle-associated protein (PVLAP), a marker for the high permeability endothelial state was observed, without significative modification of EC markers as Pecam, Cldn5 of Tjpn1. In the CNS, the canonical Wnt pathway is involved in EC to maintain BBB integrity via Wnt7a and 7b ligands. The expression of Wnt ligands was studied and that under chronic HP, a significant reduction of Wnt7b expression was reported but not of Wnt7a which may contribute to BBB stabilization.

[0108] Endothelial specific ectopic overexpression of Pdzrn3 (iECOE: inducible Endothelial Cells OverExpression) ability to accelerate vascular injury under hypoperfusion was assessed. In order to overexpress Pdzrn3 conditionally in a tissue specific manner, Pdgfb-CreER were crossed with ROSA:LNL:tTA transgenic mice (see Wang L, et al., Restricted expression of mutant SOD1 in spinal motor neurons and interneurons induces motor neuron pathology. Neurobiol Dis. 2008 March; 29(3):400-8) to generate Pdgfb-CreER/ROSA:LNL:tTA bigenic mice in which tTA was turned on specifically in EC. Then Pdgfb-CreER/ROSA:LNL:tTA mice were crossbred with TRE-Pdzrn3 mice (expressing PDZRN3-V5 and β galactosidase when tetO was activated by tTA). Both groups were obtained: Pdgfb-CreER/ROSA:LNL:tTA/tetO-Pdzrn3 triple-transgenic mice (Pdzrn3 OE), in which tTA activated tetO to express Pdzrn3 in EC and Pdgfb-CreER/ROSA:LNL:tTA (control group). For this experiment, recombination was induced with CreER and 4-hydroxytamoxifen (4HT) treatment 5 weeks after birth and animals were analyzed at 5 months. As expected, PDZRN3 and β galactosidase protein overexpression were detectable in cerebral vasculature. No modification of vessel organization in brain volumes in iECOE mice vs littermates was found by light sheet microscopy.

[0109] Data of the inventors showed that after 10 days of chronic HP, spatial recognition memory in iECOE group was even worse than those in control group (FIG. 2, right side). Chronic HP in iECOE resulted in global BBB damage with significant increase in brain microinfarct lesions, hippocampal neuronal loss (FIG. 3, right side), astrocytes activation (FIG. 4, right side) where IgG and fibrinogen extravasation into brain parenchyma in both cortical and hippocampal area were present (FIGS. 6 and 7).

[0110] Therefore, endothelial Pdzrn3 overexpression produces phenotypes opposite to those of endothelial Pdzrn3-deleted mice suggesting that PDZRN3-induced signaling tightly regulates barrier maintenance in the brain.

[0111] Loss of Pdzrn3 in endothelial cells attenuated Aβ deposit and reduces cognitive decline in a mouse model of AD.

[0112] As cerebral hypoperfusion is associated with the development of AD, it was studied whether endothelial PDZRN3 signaling contributes to worsening AD pathology. APPSwe/PSEN1dE9 mouse (APP/PS1) were crossed with transgenic Pdgfb-iCreER; Pdzrn3 to generate AD mouse cohorts deleted in endothelial cells for Pdzrn3 (APP/PS1; iECKO and their littermate control APP/PS1) and their respective non dementia (ND) littermate groups (ND-iECKO and ND-littermate controls).

[0113] A reduced cerebral blood flow was confirmed at 6 months which worsened at 12 months of age in both APP/PS1 and in APP/PS1; iECKO mouse groups. From 8 months, APP/PS1 mice were severely impaired in the spatial recognition memory paradigm whereas APP/PS1; iECKO littermates performed as well as ND-littermate control and ND-iECKO mice (FIG. 8). In APP/PS1 mouse model, amyloid plaques initially develop in the frontal cortex then spread to broad cortical and hippocampal regions. In accordance, with increasing age, APP/PS1 mice continuously accumulated more cortical and then hippocampal amyloid plaques. Strikingly, the plaque load (numbers and size) when Pdzrn3 is deleted at the age of 6 month was decreased in the cortex, and in both cortex and hippocampus at the age of 12 months in APP/PS1 mice (FIGS. 9 and 10). Abnormal activation of astrocytes and microglia has been related in human AD brains and in APP transgenic mouse model, which might increase the release of cytotoxic substances, thus causing further neuronal injury in the brain. An increase of GFAP immunolabeling at 6 months in both APP/PS1 littermates and APP/PS1; iECKO was observed; this activation was amplified at 12 months in APP/PS1 littermates but not in APP/PS1; iECKO cortical and hippocampal brain areas (see FIG. 11 for cortical area).

[0114] These findings demonstrate that the exacerbated Aβ deposits in APP/PS1 group leads to increase activation of astrocytes while deletion of Pdzrn3 in EC in reducing Aβ accumulation protects against astrogliosis.

[0115] Notably, in APP/PS1 littermate brains, large area of NeuN negative cortical cell nuclei correlated with an increase of IgG deposit at 12 months in brain sections were found; whereas NeuN negative large area in the cortex of APP/PS1; iECKO mice were not found (FIGS. 12-13). Increase extravasation of IgG in APP/PS1 brains (FIG. 13). was correlated with a rupture of BBB as reported in cortical sections from APP/PS1 triple stained for the endothelial and astrocyte markers, respectively Podocalyxin and GFAP, and with Fibrinogen to assess blood extravasation across leaky BBB while APP/PS1; Pdzrn3-deficient mice showed reduced capillary rupture.

[0116] These results suggest that deletion of Pdzrn3 in reducing extravasation of blood proteins in the cerebral parenchyma protects against neuronal death.

[0117] Pdzrn3 Induced Pathway Regulates Barrier-Specific Claudin5 and Wnt7b Gene Expression in Cerebral Vasculature

[0118] It was proposed that the level of endothelial junctional proteins decrease or relocalize in BBB dysfunction associated with CNS diseases. PDZRN3 was implicated in junctional protein polarized localization and maintain in brain EC. As accumulating evidence point to a correlation between BBB dysfunction and alteration of endothelial tight junction (TJ) complexes, whether endothelial deletion of Pdzrn3 regulates barrier specific protein expression in AD context was studied.

[0119] In intact brain vessel fragment preparation, characterization of brain vasculature in APP/PS1 mice revealed a reduced expression of Wnt7b as previously found under chronic hypoperfusion in the BCAS model; however endothelial deletion of Pdzrn3 can rescue the effect of APP/PS1 and induced Wnt7b in gliovascular units. As altered expression of Claudin 5 has been linked to a reduced Wnt canonical signaling in the CNS, the expression of endothelial cell junctional markers were studied. Endothelial deletion of Pdzrn3 induced a strong enrichment of the expression of Cldn5 both at the mRNA (FIG. 14) and protein levels in APP/PS1 mice compared to that in APP/PS1 littermate controls; in contrast, the other BBB markers such as ZO1 and occludin were not significantly modified. Confocal immune microscopy was used to characterize patterns of Claudin-5 expression at EC junctions on isolated SMA negative-capillaries. In line with our earlier finding, junctional Claudin 5 staining intensity decreased in APP/PS1 compared to that in ND-age matched control mice. Additionally, Claudin 5 was more abundant into EC-EC contact sites in brain microvessels from APP/PS1; iECKO mutant vs APP/PS1littermate control mice. The distribution of VE cadherin appeared not modified in the vessel preparations. VE cadherin was detected at equivalent levels in capillaries both from APP/PS1 vs ND-littermates and from APP/PS1-iECKO vs APP/PS1.

[0120] These data suggest that down regulation of PDZRN3 signaling leads to increase TJ strands with a reduction of vascular permeability.

[0121] These finding lends strong support to a model in which AD pathology compromises BBB Wnt pathway signalling and in which under AD context, loss of Pdzrn3 in endothelial cells maintains BBB state in a more “healthy” state.