Patent Application
20170016915
The presently disclosed subject matter relates to screening targets and compositions and methods for treatment of ciliopathy disorders.
The ciliopathies are a group of greater than 100 overlapping clinical disorders caused by defects in the primary cilium and its anchoring structure, the basal body. Since cilia are present on almost all vertebrate cell types, it is not surprising that the cilium modulates tissue and cellular events, including development, homeostasis and even cancer progression. As such, disruption of ciliary function has been shown to cause numerous human genetic disorders, including Polycystic Kidney Disease (PKD), Nephronophthisis (NPH), Bardet-Biedl Syndrome (BBS), Meckel-Gruber Syndrome (MKS), Orofaciodigital Syndrome 1 (OFD1), Joubert Syndrome (JBTS), Jeune Syndrome (JATD), Senior-Loken Syndrome (SLS) and Leber congenital amaurosis (LCA) to name but a few. Consistent with their common organelle defect, these diseases also share phenotypes, including retinal degeneration, polydactyly, cystic kidney disease, situs inversus, mental retardation, cerebellum hypoplasia, hydrometrocolpos, obesity and liver dysfunction.
Even though improved sequencing techniques have facilitated the rate of mutation identification, this group of disorders remains untreatable. So far, no efficient treatment has been designed to ameliorate symptoms or improve disease prognosis. An emerging role of primary cilium is to act as a signaling center facilitating the communication between extra- and intra-cellular signaling effector molecules during development and disease prognosis. To date, the primary cilium has been linked to several signaling pathways that are crucial for proper morphologic development, including Wnt, Shh, and Notch.
In vertebrates, the cilium and the basal body are key components of paracrine signaling transduction. This has, in turn, suggested that phenotypes of ciliopathy patients might be attributed to defective paracrine signaling (1), including polydactyly due to defective sonic hedgehog (Shh) signaling (2) and renal cysts attributed to unbalanced Wnt signaling (3). Some data have raised the possibility that a fraction of these defects, especially Wnt, are driven by nonciliary functions of ciliary and basal body proteins (2, 4); other findings have indicated that the cilium/basal body Wnt roles are likely specific to discrete spatiotemporal contexts (5-9).
Although basal body and ciliary proteins are not signaling molecules per se, these structures are thought to operate as a hub for coordinating networks of signaling cascades. Components of various signaling pathways localize to basal body and cilium (10-13). Moreover, mutations in a single basal body or ciliary gene can lead to defects in more than one signaling pathway (11, 14), while loss-of-function mutations in signaling molecules such as the Shh regulator kinesin family member 7 (KIF7) (15, 16) and the Wnt/planar cell polarity (PCP) effector Fritz (17) cause ciliopathies in some families.
Thus, there remains an unmet need for treatments to ameliorate symptoms or improve disease prognosis for ciliopathic disorders.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a ubiquitin-proteasome system (UPS)-mediated protein degradation in the presence and the absence of a candidate molecule, wherein an increase of the UPS-mediated protein degradation activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a negative regulator of a ubiquitin-proteasome system (UPS) in the presence and the absence of a candidate molecule, wherein a decrease in the activity of the negative regulator of the UPS in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a human ubiquitin peptidase (USP35) gene product or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a Zic family member 1 (ZIC1) gene product or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a small interfering RNA (siRNA) is provided. The siRNA comprises a sense RNA strand and an antisense RNA strand, wherein the sense RNA strand has a sense RNA sequence that is at least 19 nucleotides in length and at least 70% homologous to at least 19 contiguous nucleotides to of USP35 human ubiquitin peptidase cDNA sequence (SEQ ID NO: 1), and wherein the antisense RNA strand has an antisense RNA sequence that is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a short hairpin RNA (shRNA) is provided. The shRNA comprises a sense RNA sequence, an antisense RNA sequence and a hairpin sequence, wherein the sense RNA sequence is at least 19 nucleotides in length and at least 70% homologous to at least 19 contiguous nucleotides of USP35 human ubiquitin peptidase cDNA sequence (SEQ ID NO: 1), wherein the antisense RNA sequence is at least 19 nucleotides in length and complementary to the sense RNA sequence, and wherein the sense RNA sequence and the antisense RNA sequence are covalently linked by the hairpin sequence.
In one aspect of the presently disclosed subject matter a small interfering RNA (siRNA) is provided. The siRNA comprises a sense RNA strand and an antisense RNA strand, wherein the sense RNA strand has a sense RNA sequence that is from 19 to 29 nucleotides in length and at least 70% homologous to at least 19 contiguous nucleotides of ZIC1 Zic family member 1 cDNA sequence (SEQ ID NO: 2), and wherein the antisense RNA strand has an antisense RNA sequence that is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a short hairpin RNA (shRNA) is provided. The shRNA comprises a sense RNA sequence, an antisense RNA sequence and a hairpin sequence, wherein the sense RNA sequence is at least 19 nucleotides in length and at least 70% homologous to at least 19 contiguous nucleotides of ZIC1 Zic family member 1 cDNA sequence (SEQ ID NO: 2), wherein the antisense RNA sequence is at least 19 nucleotides in length and complementary to the sense RNA sequence, and wherein the sense RNA sequence and the antisense RNA sequence are covalently linked by the hairpin sequence.
In one aspect of the presently disclosed subject matter a method is provided for treating a ciliopathy disorder, the method includes administering at least one small interfering RNA (siRNA), comprising a sense RNA sequence and an antisense RNA sequence, to an individual having a ciliopathy disorder; and monitoring the level of the ciliopathy disorder, wherein the sense RNA sequence is at least about 70% homologous to at least 19 contiguous nucleotides of a USP35 ubiquitin peptidase cDNA sequence (SEQ ID NO: 1) and wherein the antisense RNA sequence is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a method is provided for treating a ciliopathy disorder, the method includes administering at least one short hairpin RNA (shRNA), comprising a sense RNA sequence and an antisense RNA sequence covalently linked by a hairpin sequence to an individual having a ciliopathy disorder; and monitoring the level of the ciliopathy disorder, wherein the sense RNA sequence is at least about 70% homologous to at least 19 contiguous nucleotides of a USP35 ubiquitin peptidase cDNA sequence (SEQ ID NO: 1) and wherein the antisense RNA sequence is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a method is provided for treating a ciliopathy disorder, the method includes administering at least one small interfering RNA (siRNA), comprising a sense RNA sequence and an antisense RNA sequence, to an individual having a ciliopathy disorder; and monitoring the level of the ciliopathy disorder, wherein the sense RNA sequence is at least about 70% homologous to at least 19 contiguous nucleotides of a ZIC1 Zic family member 1 cDNA sequence (SEQ ID NO: 2) and wherein the antisense RNA sequence is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a method is provided for treating a ciliopathy disorder, the method includes administering at least one small hairpin RNA (shRNA), comprising a sense RNA sequence and an antisense RNA sequence covalently linked by a hairpin sequence, to an individual having a ciliopathy disorder; and monitoring the level of the ciliopathy disorder, wherein the sense RNA sequence is at least about 70% homologous to at least 19 contiguous nucleotides of a ZIC1 Zic family member 1 cDNA sequence (SEQ ID NO: 2) and wherein the antisense RNA sequence is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes determining the ability of a candidate molecule to rescue a defect in a cell-based or an animal-based model, wherein the cell-based or the animal-based model comprises a silenced, reduced, or depleted expression of one or more ciliary genes comprising a BBS4 (Bardet-Biedl syndrome 4) gene, a BBS1 (Bardet-Biedl syndrome 1) gene, or an OFD1 (Oral-facial-digital syndrome 1) gene that results in the defect, and wherein the ability of the candidate molecule to rescue the defect identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
One aspect of the present disclosure provides a method of treating a ciliopathy disorder in a subject comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a proteasome agonist such that the ciliopathy disorder is treated.
The foregoing aspects and other features of the invention are explained in the following description, taken in connection with the accompanying drawings.
For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications to of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
The presently disclosed subject matter provides a common mechanism for the multiple divergent pathways for ciliary and basal body protein involvement that have been shown to be associated with signaling defects. In one aspect of the present disclosure, the ubiquitin-proteasome system (UPS) is provided as a common mechanism and a target for treatment of ciliopathy disorders. First, it was noted that the basal body is known to be a proteolytic center (18-21). Second, it was noted that previous reports indicate that disruption of some basal body proteins result in loss of proteasome-dependent degradation of β-catenin (5), a phenotype reproduced subsequently (22). Finally, it was noted that, in addition to Wnt, proteasomal degradation is implicated in most paracrine signaling cascades known to be defective in basal body mutants.
For example, in Notch-mediated lateral inhibition, a transmembrane ligand of the Delta-Serrate-LAG2 (DSL) family binds a transmembrane Notch receptor in the adjacent target cells (23). To reduce Notch signaling, DSL ligands are ubiquitinated, internalized, and degraded by the proteasome (24); similarly, the intracellular domain of the Notch receptor is degraded by the proteasome to reduce Notch signaling (25). Likewise, when Shh ligand is present, glioma-associated oncogenes 2 and 3 (GLI2/3) exist in their full-length activator forms, whereas they are truncated by proteasome-mediated proteolysis to their repressor forms when Shh is removed (26); both the activator and repressor forms of GLI2/3 are also degraded by the proteasome (27). Besides GLI2/3, suppressor of fused homolog (SUFU), a negative regulator of Shh signaling that physically localizes at the cilium, is also degraded in a proteasome-dependent manner (28). Aspects of the present disclosure provide that basal body and ciliary proteins can regulate multiple signaling pathways by controlling proteasome-mediated degradation of signaling mediators.
Aspects of the presently disclosed subject matter demonstrate that suppression of basal body-localized ciliopathy proteins can lead to defective proteasomal degradation of such mediators, which in turn can cause dysfunction in three major cilia-associated signaling pathways (Shh, Wnt, and Notch) in vitro and in vivo. These results are unlikely to reflect nonspecific cellular malaise; as not only could the ciliopathy proteins tested herein interact with specific regulatory subunits of the proteasome holoenzyme, but also depletion of each of the ciliopathy proteins Bardet-Biedl syndrome 4 (BBS4) and oral-facial-digital syndrome 1 (OFD1) selectively perturbed the subunit composition of the centrosomal proteasome.
More specifically, the presently disclosed subject matter demonstrates that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In wild-type (WT) cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice.
In addition, the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins was tested in the presently disclosed subject matter. It was discovered that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, these data indicate that basal body proteasomal regulation can govern paracrine signaling pathways and indicate that augmenting proteasomal function can benefit ciliopathy patients.
Furthermore, in a separate approach from the experiments described above, a genome-wide siRNA screening assay was performed in a human cell line model of loss of ciliary function to identify genes whose suppression can rescue the aberrant signaling transduction caused by loss of ciliary function. The siRNA screening assay and the resulting data are described in the present disclosure. In this siRNA assay, USP35, a deubiquitinase (ubiquitin peptidase) that acts as a negative regulator of the ubiquitin-proteasome-system (UPS), was identified, as well as 12 other gene targets for ciliopathy disorders. The veracity of the observed rescue effect seen in the cell model was then further tested in an animal model of ciliopathy disorders for 6 of the 13 identified targets. Specifically, a zebrafish model was used to recapitulate pathogenic features in patients. Depletion of the gene product for 5 of the 6 identified targets in the zebrafish model, including USP35, was demonstrated to ameliorate the cerebellar and renal defects in the ciliary deficient animal model.
Thus, one aspect of the present disclosure provides methods for benefiting ciliopathy patients by increasing UPS-mediated protein degradation activity.
Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
As used herein, the term “ciliopathy disorder”, “ciliopathy disease”, “ciliopathic disease”, “ciliopathies” and ciliopathic disorders” are used interchangeably and refer to those genetic disorders of the cellular cilia, the cilia anchoring structures, the basal bodies, and/or ciliary function. Examples of such disorder include, but are not limited to, Alstrom Syndrome, Bardet-Biedl Syndrome (BBS) (e.g., BBS1, BBS2, BBS4, BBS5, BBS7, BBS9, BBS10, BBS12, ARL6, MKKS, TTC8, TRIM32), Joubert Syndrome, Meckel-Gruber syndrome, Nephronophthisis, Oral-facial-digital syndrome 1 (OFD1), Senior-Loken Syndrome, Polycystic kidney disease, primary ciliary dyskinsesia, asphyxiating thoracic dysplasia, Marden-Walker syndrome, situs inversus/Isomerism, and the like.
As used herein the term “ubiquitin-proteasome system (UPS)” is used interchangeably with the term “ubiquitin-proteasome pathway” or the term “proteasome pathway”. As used herein the term “proteasome agonist” refers to any compound or molecule that is capable of activating the proteasome pathway, either by activating the proteasome itself, or a protein within or associated with the proteasome pathway that results in the activation of the proteasome. In one example a protein that is within or associated with the proteasome pathway is a negative regulator of the proteosome pathway. Examples of proteosome agonists include, but are not limited to, sulforaphane (SFN; 1-isothiocyanato-4(R)-methylsulfinylbutane) and mevalonolactone (MVA).
As used herein, the term “subject” and “patient” and “individual” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, mice, chickens, amphibians, reptiles, and the like. Preferably, the subject is a human patient. More preferably, the subject is a human patient suffering from a ciliopathy disorder.
As used herein, “treatment” is a clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition. “Treatments” refer to one or both of therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already affected by a disease or disorder or undesired physiological condition as well as those in which the disease or disorder or undesired to physiological condition is to be prevented.
The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a ubiquitin-proteasome system (UPS)-mediated protein degradation in the presence and the absence of a candidate molecule, wherein an increase of the UPS-mediated protein degradation activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder. The UPS-mediated protein degradation activity can be measured directly at the ubiquitin-proteasome holoenzyme. The candidate molecule can include a small molecule or an antibody.
The UPS-mediated protein degradation activity can be measured indirectly via a cell-based or an animal-based model, wherein the cell-based or the animal-based model has a silenced, reduced, or depleted expression of one or more ciliary genes having a BBS4 (Bardet-Biedl syndrome 4) gene, a BBS1 (Bardet-Biedl syndrome 1) gene, or an OFD1 (Oral-facial-digital syndrome 1) gene that results in reduced ubiquitin-proteasome system (UPS)-mediated protein degradation. The candidate molecule can include a small molecule, an antibody, a RNA interference molecule (RNAi), a short hairpin RNA (shRNA), or a small interfering RNA (siRNA).
The cell-based model can include a human retinal pigmentosa epicedium cell line (RPE). The ciliary gene can be the BBS4 gene. The RPE cell line can stably expresses a short hairpin RNA (shRNA) against BBS4 expression.
The animal-based model can include a morphant zebrafish embryo model having a bbs4 depletion-induced convergent extension (CE) defect, a cerebellum organizational abnormality, and a renal development abnormality. The animal-based model can include an Ofd1 knockout mouse model.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a negative regulator of a ubiquitin-proteasome system (UPS) in the presence and the absence of a candidate molecule, wherein a decrease in the activity of the negative regulator of the UPS in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
The negative regulator of the ubiquitin-proteasome system (UPS) can include: a ubiquitin peptidase (USP35) gene product or an ortholog thereof, a Zic family member 1 (ZIC1) gene product or an ortholog thereof, a dopamine receptor D5 (DRD5) gene product or an ortholog thereof, a prothymosin alpha gene sequence 28 (PTMA) or an ortholog thereof, an endo-beta-N-acetylglucosaminidase (ENGASE) gene product or an ortholog thereof, a phosphatidylinositol transfer protein (PITPNM2) gene product or an ortholog thereof, a Rhox homeobox family member 1 (RHOXF1) gene product or an ortholog thereof, an ectonucleoside triphosphate diphosphohydrolase 6 (ENTPD6) gene product or an ortholog thereof, a chromosome 14 open reading frame 166 (C14orf166) gene product or an ortholog thereof, a cleavage and polyadenylation factor subunit homolog (PCF11) gene product or an ortholog thereof, a testis expressed 36 (TEX36) gene product or an ortholog thereof, or a tudor domain containing 12 (TDRD12) gene product or an ortholog thereof.
In the method for screening for a therapeutic molecule to treat a ciliopathy disorder, the candidate molecule can include a small molecule, an antibody, a RNA interference molecule (RNAi), a short hairpin RNA (shRNA), or a small interfering RNA (siRNA).
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a ubiquitin peptidase USP35 gene product, or an ortholog to thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a Zic family member 1 (ZIC1) gene product or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of an endo-beta-N-acetylglucosaminidase (ENGASE) gene product, or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a membrane-associated 2 phosphatidylinositol transfer protein (PITPNM2) gene product, or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a dopamine receptor D5 (DRD5) gene product or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a Rhox homeobox family member 1 (RHOXF1) gene product or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of an ectonucleoside triphosphate diphosphohydrolase 6 (ENTPD6) gene product or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a chromosome 14 open reading frame 166 (C14orf166) gene product or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a cleavage and polyadenylation factor subunit homolog (PCF11) gene product or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a testis expressed 36 (TEX36) gene product or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes measuring the activity of a tudor domain containing 12 (TDRD12) gene product or an ortholog thereof, in the presence and the absence of a candidate molecule, wherein a decrease of the activity in the presence of the candidate molecule identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In one aspect of the presently disclosed subject matter a small interfering RNA (siRNA) is provided. The siRNA comprises a sense RNA strand and an antisense RNA strand, wherein the sense RNA strand has a sense RNA sequence that is at least 19 nucleotides in length and at least 70% homologous to at least 19 contiguous nucleotides of USP35 human ubiquitin peptidase cDNA sequence (SEQ ID NO: 1), and wherein the antisense RNA strand has an antisense RNA sequence that is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a short hairpin RNA (shRNA) is provided. The shRNA comprises a sense RNA sequence, an antisense RNA sequence and a hairpin sequence, wherein the sense RNA sequence is at least 19 nucleotides in length and at least 70% homologous to at least 19 contiguous nucleotides of USP35 human ubiquitin peptidase cDNA sequence (SEQ ID NO: 1), wherein the antisense RNA sequence is at least 19 nucleotides in length and complementary to the sense RNA sequence, and wherein the sense RNA sequence and the antisense RNA sequence are covalently linked by the hairpin sequence.
In one aspect of the presently disclosed subject matter a small interfering RNA (siRNA) is provided. The siRNA comprises a sense RNA strand and an antisense RNA strand, wherein the sense RNA strand has a sense RNA sequence that is from 19 to 29 nucleotides in length and at least 70% homologous to at least 19 contiguous nucleotides of ZIC1 Zic family member 1 cDNA sequence (SEQ ID NO: 2), and wherein the antisense RNA strand has an antisense RNA sequence that is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a short hairpin RNA (shRNA) is provided. The shRNA comprises a sense RNA sequence, an antisense RNA sequence and a hairpin sequence, wherein the sense RNA sequence is at least 19 nucleotides in length and at least 70% homologous to at least 19 contiguous nucleotides of ZIC1 Zic family member 1 cDNA sequence (SEQ ID NO: 2), wherein the antisense RNA sequence is at least 19 nucleotides in length and complementary to the sense RNA sequence, and wherein the sense RNA sequence and the antisense RNA sequence are covalently linked by the hairpin sequence.
In one aspect of the presently disclosed subject matter a small interfering RNA (siRNA) is provided. The siRNA comprises a sense RNA strand and an antisense RNA strand, wherein the sense RNA strand has a sense RNA sequence that is at least 19 nucleotides in length, wherein the antisense RNA strand has an antisense RNA sequence that is complementary to the sense RNA sequence, and wherein the sense RNA sequence comprises: at least 70% homology to at least 19 contiguous nucleotides of a DRD5 dopamine receptor D5 cDNA sequence (SEQ ID NO: 3); at least 70% homology to at least 19 contiguous nucleotides of a PTMA prothymosin alpha gene sequence 28 cDNA sequence (SEQ ID NO: 4); at least 70% homology to at least 19 contiguous nucleotides of a ENGASE endo-beta-N-acetylglucosaminidase cDNA sequence (SEQ ID NO: 5); at least 70% homology to at least 19 contiguous nucleotides of a PITPNM2 phosphatidylinositol transfer protein cDNA sequence (SEQ ID NO: 6); at least 70% homology to at least 19 contiguous nucleotides of a RHOXF1 Rhox homeobox family member 1 cDNA sequence (SEQ ID NO: 7); at least 70% homology to at least 19 contiguous nucleotides of a ENTPD6 ectonucleoside triphosphate diphosphohydrolase 6 cDNA sequence (SEQ ID NO: 8); at least 70% homology to at least 19 contiguous nucleotides of a C14orf166 chromosome 14 open reading frame 166 cDNA sequence (SEQ ID NO: 9); at least 70% homology to at least 19 contiguous nucleotides of a PCF11 cleavage and polyadenylation factor subunit homolog cDNA sequence (SEQ ID NO: 10); at least 70% homology to at least 19 contiguous nucleotides of a TEX36 testis expressed 36 cDNA sequence (SEQ ID NO: 11); or at least 70% homology to at least 19 contiguous nucleotides of a TDRD12 tudor domain containing 12 cDNA sequence (SEQ ID NO: 12).
In one aspect of the presently disclosed subject matter a short hairpin RNA (shRNA) is provided. The shRNA comprises a sense RNA sequence, an antisense RNA sequence and a hairpin sequence, wherein the sense RNA sequence is at least 19 nucleotides in length, wherein the antisense RNA sequence is at least 19 nucleotides in length and complementary to the sense RNA sequence, wherein the sense RNA sequence and the antisense RNA sequence are covalently linked by the hairpin sequence, and wherein the sense RNA sequence comprises: at least 70% homology to at least 19 contiguous nucleotides of a DRD5 dopamine receptor D5 cDNA sequence (SEQ ID NO: 3); at least 70% homology to at least 19 contiguous nucleotides of a PTMA prothymosin alpha gene sequence 28 cDNA sequence (SEQ ID NO: 4); at least 70% homology to at least 19 contiguous nucleotides of a ENGASE endo-beta-N-acetylglucosaminidase cDNA sequence (SEQ ID NO: 5); at least 70% homology to at least 19 contiguous nucleotides of a PITPNM2 phosphatidylinositol transfer protein cDNA sequence (SEQ ID NO: 6); at least 70% homology to at least 19 contiguous nucleotides of a RHOXF1 Rhox homeobox family member 1 cDNA sequence (SEQ ID NO: 7); at least 70% homology to at least 19 contiguous nucleotides of a ENTPD6 ectonucleoside triphosphate diphosphohydrolase 6 cDNA sequence (SEQ ID NO: 8 at least 70% homology to at least 19 contiguous nucleotides of a C14orf166 chromosome 14 open reading frame 166 cDNA sequence (SEQ ID NO: 9); at least 70% homology to at least 19 contiguous nucleotides of a PCF11 cleavage and polyadenylation factor subunit homolog cDNA sequence (SEQ ID NO: 10); at least 70% homology to at least 19 contiguous nucleotides of a TEX36 testis expressed 36 cDNA sequence (SEQ ID NO: 11); or at least 70% homology to at least 19 contiguous nucleotides of a TDRD12 tudor domain containing 12 cDNA sequence (SEQ ID NO: 12).
In one aspect of the presently disclosed subject matter methods are provided for treating a ciliopathy disorder by administering a small interfering RNA (siRNA) or a short hairpin RNA (shRNA) to an individual having a ciliopathy disorder. The siRNA's and shRNA's can be administered as a pharmaceutical composition in combination with a pharmaceutically acceptable carrier. The siRNA's and shRNA's can be administered in combination with a delivery reagent.
In one aspect of the presently disclosed subject matter a method is provided for treating a ciliopathy disorder, the method includes administering at least one small interfering RNA (siRNA), comprising a sense RNA sequence and an antisense RNA sequence, to an individual having a ciliopathy disorder; and monitoring the level of the ciliopathy disorder, wherein the sense RNA sequence is at least about 70% homologous to at least 19 contiguous nucleotides of a USP35 ubiquitin peptidase cDNA sequence (SEQ ID NO: 1) and wherein the antisense RNA sequence is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a method is provided for treating a ciliopathy disorder, the method includes administering at least one short hairpin RNA (shRNA), comprising a sense RNA sequence and an antisense RNA sequence covalently linked by a hairpin sequence to an individual having a ciliopathy disorder; and monitoring the level of the ciliopathy disorder, wherein the sense RNA sequence is at least about 70% homologous to at least 19 contiguous nucleotides of a USP35 ubiquitin peptidase cDNA sequence (SEQ ID NO: 1) and wherein the antisense RNA sequence is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a method is provided for treating a ciliopathy disorder, the method includes administering at least one small interfering RNA (siRNA), comprising a sense RNA sequence and an antisense RNA sequence, to an individual having a ciliopathy disorder; and monitoring the level of the ciliopathy disorder, wherein the sense RNA sequence is at least about 70% homologous to at least 19 contiguous nucleotides of a ZIC1 Zic family member 1 cDNA sequence (SEQ ID NO: 2) and wherein the antisense RNA sequence is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a method is provided for treating a ciliopathy disorder, the method includes administering at least one small hairpin RNA (shRNA), comprising a sense RNA sequence and an antisense RNA sequence covalently linked by a hairpin sequence, to an individual having a ciliopathy disorder; and monitoring the level of the ciliopathy disorder, wherein the sense RNA sequence is at least about 70% homologous to at least 19 contiguous nucleotides of a ZIC1 Zic family member 1 cDNA sequence (SEQ ID NO: 2) and wherein the antisense RNA sequence is complementary to the sense RNA sequence.
In one aspect of the presently disclosed subject matter a method is provided for treating a ciliopathy disorder, the method includes administering at least one small interfering RNA (siRNA), comprising a sense RNA sequence and an antisense RNA sequence, and wherein the antisense RNA sequence is complementary to the sense RNA sequence, to an individual having a ciliopathy disorder; and monitoring the level of the ciliopathy disorder, wherein the sense RNA sequence is at least about 70% homologous to at least 19 contiguous nucleotides of a cDNA sequence comprising: a cDNA sequence comprising a DRD5 dopamine receptor D5 cDNA sequence (SEQ ID NO: 3); a cDNA sequence comprising a PTMA prothymosin alpha gene sequence 28 cDNA sequence (SEQ ID NO: 4); a cDNA sequence comprising an ENGASE endo-beta-N-acetylglucosaminidase cDNA sequence (SEQ ID NO: 5); a cDNA sequence comprising a PITPNM2 phosphatidylinositol transfer protein cDNA sequence (SEQ ID NO: 6); a cDNA sequence comprising a RHOXF1 Rhox homeobox family member 1 cDNA sequence (SEQ ID NO: 7); a cDNA sequence comprising an ENTPD6 ectonucleoside triphosphate diphosphohydrolase 6 cDNA sequence (SEQ ID NO: 8); a cDNA sequence comprising a C14orf166 chromosome 14 open reading frame 166 cDNA sequence (SEQ ID NO: 9); a cDNA sequence comprising a PCF11 cleavage and polyadenylation factor subunit homolog cDNA sequence (SEQ ID NO: 10); a cDNA sequence comprising a TEX36 testis expressed 36 cDNA sequence (SEQ ID NO: 11); or a cDNA sequence comprising a TDRD12 tudor domain containing 12 cDNA sequence (SEQ ID NO: 12).
In one aspect of the presently disclosed subject matter a method is provided for treating a ciliopathy disorder, the method includes administering at least one short hairpin RNA (shRNA), comprising a sense RNA sequence and an antisense RNA sequence covalently linked by a hairpin sequence, and wherein the antisense RNA sequence is complementary to the sense RNA sequence, to an individual having a ciliopathy disorder; and monitoring the level of the ciliopathy disorder, wherein the sense RNA sequence is at least about 70% homologous to at least 19 contiguous nucleotides of a cDNA sequence comprising: a cDNA sequence comprising a DRD5 dopamine receptor D5 cDNA sequence (SEQ ID NO: 3); a cDNA sequence comprising a PTMA prothymosin alpha gene sequence 28 cDNA sequence (SEQ ID NO:4); a cDNA sequence comprising an ENGASE endo-beta-N-acetylglucosaminidase cDNA sequence (SEQ ID NO: 5); a cDNA sequence comprising a PITPNM2 phosphatidylinositol transfer protein cDNA sequence (SEQ ID NO: 6); a cDNA sequence comprising a RHOXF1 Rhox homeobox family member 1 cDNA sequence (SEQ ID NO: 7); a cDNA sequence comprising an ENTPD6 ectonucleoside triphosphate diphosphohydrolase 6 cDNA sequence (SEQ ID NO: 8); a cDNA sequence comprising a C14orf166 chromosome 14 open reading frame 166 cDNA sequence (SEQ ID NO: 9); a cDNA sequence comprising a PCF11 cleavage and polyadenylation factor subunit homolog cDNA sequence (SEQ ID NO: 10); a cDNA sequence comprising a TEX36 testis expressed 36 cDNA sequence (SEQ ID NO: 11); or a cDNA sequence comprising a TDRD12 tudor domain containing 12 cDNA sequence (SEQ ID NO: 12).
In one aspect of the presently disclosed subject matter a method is provided for screening for a therapeutic molecule to treat a ciliopathy disorder. The method includes determining the ability of a candidate molecule to rescue a defect in a cell-based or an animal-based model, wherein the cell-based or the animal-based model comprises a silenced, reduced, or depleted expression of one or more ciliary genes comprising a BBS4 (Bardet-Biedl syndrome 4) gene, a BBS1 (Bardet-Biedl syndrome 1) gene, or an OFD1 (Oral-facial-digital syndrome 1) gene that results in the defect, and wherein the ability of the candidate molecule to rescue the defect identifies the candidate molecule as a potential therapeutic molecule to treat a ciliopathy disorder.
In the method for screening for a therapeutic molecule to treat a ciliopathy disorder the candidate molecule can include a small molecule, an antibody, a RNA interference molecule (RNAi), a short hairpin RNA (shRNA), or a small interfering RNA (siRNA).
The cell-based model can include a human retinal pigmentosa epicedium cell line (RPE). The ciliary gene can include the BBS4 gene. The human retinal pigmentosa epicedium cell line can stably expresses a short hairpin RNA (shRNA) against BBS4 expression. The defect can be hyper activation of the Wnt/3-cat signaling. In the RPE cell line based model, the candidate molecule can be a RNA interference molecule (RNAi), a short hairpin RNA (shRNA), or a small interfering RNA (siRNA), and determining the ability of the candidate molecule to rescue the defect can include transfecting the RPE cell line with the candidate molecule.
The animal-based model can include a morphant zebrafish embryo model, wherein the defect comprises a bbs4 depletion-induced convergent extension (CE) defect, a cerebellum organizational abnormality, and a renal development abnormality. In the morphant zebrafish embryo model, the candidate molecule can include a RNA interference molecule (RNAi), a short hairpin RNA (shRNA), or a small interfering RNA (siRNA), and the determining the ability of the candidate molecule to rescue the defect can include injecting the zebrafish embryo model with the candidate molecule.
The animal-based model can include an Ofd1 knockout mouse model.
One aspect of the present disclosure provides a method of treating a ciliopathic disorder in a subject comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a proteasome agonist such that the ciliopathic disorder is treated.
In some embodiments, the proteasome agonist comprises SFN. In other embodiments, the proteasome agonist comprises MVA.
Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. It is shown in the presently provided disclosure that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. In addition, it is demonstrated in the presently provided disclosure provided below that overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Furthermore, it is demonstrated in the presently provided disclosure that other proteasome-dependent pathways not known to be associated with ciliopathies are similarly defective in the absence of ciliopathy proteins. More specifically, it was found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, these data indicate that basal body proteasomal regulation can govern paracrine signaling pathways.
Accumulation of signaling mediators upon depletion of ciliopathy proteins. It has been reported previously that perturbation of some ciliopathy proteins alters the stability of Wnt signaling mediators such as β-catenin and Dishevelled (5, 8, 21, 29, 30). To study the physiological relevance of these findings in vivo, Bbs4−/− mice were mated with a transgenic proteasome reporter mouse line expressing unstable ubiquitin-tagged Green Fluorescent Protein (GFP) (31) to generate UbG76V-Gfp Bbs4−/− mice. As a hallmark of potential proteasomal dysfunction, GFP levels were examined in a broad range of tissues with known pathology in BBS and no aberrant GFP accumulation was observed in the kidney and liver of UbG76V-Gfp Bbs4−/− mice (
Given the specificity of GFP degradation defects in the photoreceptors of Bbs4−/− mice and previous data on the abnormal stability of β-catenin in BBS4-depleted cells (5), the question was asked whether proteasomal dysfunction might be more broadly relevant to basal body-mediated paracrine signal regulation. If this is true, then: (a) other basal body proteins should have similar functions; and (b) other known basal body-mediated pathways should also be perturbed.
To evaluate functional links between basal body proteins, an in silico study was conducted ato analyze a network of coexpressed basal body and ciliary genes through a publicly available online tool (http://netview.tigem.it), which reconstructs the regulatory interactions among genes from genome scale measurements of gene expression profiles (32). This analysis revealed a subset of centrosomal transcripts (P=3.5×10−7) including BBS1, BBS4, and OFD1 (
The established cilium-dependent paracrine signaling pathway, Hedgehog signaling (Hh), was used (2). In addition, an in vitro neural differentiation model was developed, in which the embryonic WT murine stem cells (WTESs) and Ofd1-deficient murine embryonic stem cells (KOESs) were cultured to generate neural progenitors (
Similar to Hh and Wnt signaling, Notch signaling is regulated through the cilium (13), and if the overarching hypothesis is correct, this pathway should also be perturbed in the mutants produced as described herein. As a first test, Flag-tagged Notch1 intracellular domain (NICD) was cotransfected into HEK-293-FT cells depleted of BBS4 by shRNAmediated gene silencing (pSuperBBS4) (5). Immunoblot analysis of transfected cells revealed an elevation of NICD protein levels upon knockdown of BBS4 (
Selective Disruption of UPS Caused by Loss of Ciliopathy Proteins.
Given the data on Wnt, Shh, and Notch and the fact that the processing of components from these paracrine signaling pathways is known to be mediated by the proteasome (11, 35), the question was asked whether proteasomal agonists could ameliorate the signaling phenotypes of basal body mutants.
The expression levels of three catalytic subunits of the proteasome, proteasome subunit 13 types 5, 6, and 7 (PSMB5, PSMB6, and PSMB7), as well as the corresponding peptidase activities, increase upon treatment with sulforaphane (36) [SFN; 1-isothiocyanato-4(R)-methylsulfinylbutane], an isothiocyanate extracted from cruciferous vegetables, rendering this compound an attractive initial substrate for the studies. It was found that treatment with SFN rescued the accumulation of GLI2FL, upon overexpressing the proteins of interest. Therefore, BBS4 was overexpressed in cells and a depletion was observed of both NICD (leucinyl
leucinyl
norleucinal (MG132) and lactacystin as well as DMSO vehicle as a control. While cells overexpressing BBS4 showed a 21%-50% reduction in total NICD levels (
Regulation of the Proteasome Complex by Ciliopathy Proteins.
Although the presently disclosed observations in mouse mutants, zebrafish embryos (5), and cultured cells argue that these basal body proteins are responsible for regulating the proteasome, the mechanism of that action is unclear, leaving the possibility that the observed phenotypes are surrogate effects of generalized GLI3FL, and SUFU in T8-derived Ofd1KO neurons (
Taken together, these data argue for a role of basal body proteins in regulating proteasome-mediated degradation and that a reciprocal signaling/proteasomal phenotype cellular “malaise” should be observed. Therefore the possibility was explored of direct biochemical relationships between BBS1, BBS4, and OFD1 and the proteasome.
First, an unbiased mass spectrometry screen was conducted to identify putative OFD1-interacting proteins. This experiment uncovered a spectrum of proteasomal subunits including regulatory proteasome ATPase subunit 6 (RPT6), a finding confirmed by semiendogenous coimmunoprecipitation (
The fraction of 26S-bound RPN10 in BBS4-depleted cells was also assayed and a modest but significant reduction (20%±3%) in RPN10 was observed (
Activation of Proteasomal Components Ameliorates Signaling Defects Caused by Loss of Ciliopathy Proteins that Reside at the Basal Body.
These observations next led to speculation that (a) if reduction of proteasomal subunits in the centrosomal fraction of BBS4 or OFD1-depleted cells results in hampered degradation of signaling mediators, then increasing expression of the corresponding proteasomal subunits may ameliorate defective signaling phenotypes; and (b) activation of the proteasome might compensate for signaling defects.
It has been reported previously that loss of ciliopathy proteins produces defects in convergent extension (CE) movements during gastrulation in zebrafish embryos (5, 29, 30). In addition to and independent of these phenotypes, defects were observed in the present disclosure in the definition of somite boundaries in zebrafish embryos injected with morpholinos against bbs genes (bbs morphants [bbs MO]) (
Upon injection of the established bbs4 morpholino (5, 38) into 1 to 8-cell-stage embryos, and scoring at the 9±1 somite stage (9±1 ss), 92% of embryos exhibited an expansion of the anteroposterior midline expression domain of her4 (
Given the availability of experimentally tractable signaling phenotypes, the question was asked whether overexpression of proteasomal subunits that were seen to mislocalize in BBS4 and OFD1-depleted cells might rescue the bbs4 and ofd1 morphant phenotype. Upon blind scoring for the effects of coinjecting human RPN10, RPN13, or RPT6 mRNA with the bbs4 or ofd1 morpholino, substantial rescue of defective CE, somitic definition, and persistent her4 expression was measured (
Next, as an independent test, bbs4 morphants coinjected with SFN, a known transcriptional activator of proteasomal subunits (36) were examined. Upon blind scoring at the 9±1 ss, there was a significant reduction of bbs4 morphants with CE and somitic defects (P<0.001;
To substantiate the rescue effects of SFN, another proteasomal agonist was used, mevalonolactone [known as mevalonic acid lactone, mevalonate, and (±)-β-hydroxy-β-methyl-δ-valerolactone and abbreviated hereafter as MVA], to ask whether broad activation of the proteasome can alleviate basal body-dependent phenotypes. MVA was coinjected with the bbs4 morpholino into zebrafish embryos, and upon blind scoring at 9±1 ss, a reduction in bbs4 morphant zebrafish with CE defects from 47.8% to 11.4% was found with MVA (
NF-κB Signaling Defects in Basal Body Ciliopathy Mutants can be Rescued by Activation of the Proteasome.
The presently disclosed findings indicate that Wnt, Notch, and Shh phenotypes generated upon loss of three basal body proteins might converge at the to point of proteasomal degradation. Therefore a model was considered in which the basal body region serves as a broad regulator of paracrine signaling through proteasomal degradation. If this model is true, then (a) other paracrine pathways not implicated previously in ciliary/basal body biology, but known to be regulated by the proteasome, should exhibit signaling defects upon depletion of BBS proteins or OFD1; and (b) the model should also be able to predict the direction of the phenotype (suppressed or excessive signaling). To test this, NF-κB signaling was used, a pathway involved in inflammatory responses and lymphoid organogenesis with no known link to basal body (dys)function. In response to stimuli, IκBs are degraded by the proteasome, releasing NF-κB to translocate to the nucleus and activate the transcription of target genes (41).
HEK-293-FT cells were transfected with an NF-κB luciferase reporter plasmid containing three copies of the KB response elements of the murine MHC class I promoter (3X-κB-L). Cells stimulated by TNF-α and cotransfected with the pSuperBBS4, pSuperBBS1, and pSuperOFD1 plasmids displayed a 55%, 53%, and 72% reduction in NF-κB activity, respectively, compared with that of control cells; incubation with SFN for 6 hours restored NF-κB activity (
Finally, to probe the potential physiological relevance of the observed NF-κB data in vivo, the expression and protein levels of the proteasomal substrate IκBβ in mice lacking Ofd1 was examined. Immunoblot analysis showed no differences in IκBβ protein levels on protein lysates from E10.5 Ofd1Δ4-5/y mutant embryos and controls (data not shown). Since loss of Ofd1 in mice results in prenatal lethality, Ofd1-f loxed mice (Ofd1fl) were generated and crossed with a CAG-Cre-ER™-inducible general deletor line to examine IκBβ protein levels in postnatal Ofd1 knockout mice. In Ofd1fl/y CAG-Cre-ER™ (Ofd1fl/y CKO) mice, Ofd1 inactivation was achieved at E18.5 by tamoxifen injection, and renal cysts were not observed at P8 (precystic stage). By P20, a replacement was observed of the renal parenchyma by cysts (cystic stage). Immunoblot analysis revealed an accumulation of IκBβ in protein lysates from kidney of Ofd1fl/y CKO male mice compared with controls; crucially, this phenotype was evident in both precystic and cystic stages (
The study of ciliary and basal body proteins has highlighted a complex role for this cellular region in the regulation of signaling pathways. These observations have raised critical questions, including whether dedicated signaling transduction machinery aggregate around the cilium and basal body. Several transduction components have been localized to the basal body and/or the ciliary axoneme, including Smoothened, GLI proteins, SUFU, β-catenin, adenomatosis polyposis coli (APC), and Notch3 (8, 10, 11, 13, 29). At the same time, a model in which the cilium facilitates each pathway independently is difficult to reconcile with the fact that (a) with the possible exception of the PCP transducer Fritz (17) and the Shh motor KIF7 (15, 16), none of the other 60-plus genes and proteins mutated in human ciliopathies are components of a specific paracrine signaling pathway; (b) the phenotype of ciliopathy patients is a mixture of defects more consistent with a context-specific pathway dysfunction; and (c) animal models ablated for specific ciliary or basal body proteins exhibit multiple signaling defects (1).
Without being limited to any one particular mechanism, the data presented herein suggest a simpler model, in which at least some basal body proteins play a role in signal transduction regulation by exerting their primary effect not on a given pathway per se, but by regulating context-dependent proteolytic degradation. The alternative would be that the observed phenotypes are the nonspecific consequence of generalized cellular malaise and that the observed rescue effects were reflective of broad improvement in the ability of the cell to eliminate proteins targeted for degradation. Taken together, the experiments provided herein favor the former model. Cells and embryos suppressed or ablated for each of BBS1, BBS4, and OFD1 had defects in proteasomal clearance of both reporter proteins and specific signaling components that included β-catenin, NICD, JAG1, GLI2, GLI3, SUFU, and IκBβ. It is also notable that the pronounced defects in proteasomal activity were concomitant with overt anatomical pathology in the murine models of disease described herein, such as in the retina of Bbs mice and the cystic kidneys of conditional Ofd1 animals, whereas no GFP accumulation was observed in Bbs mutant kidneys that, in the colony described herein, never exhibited cyst formation.
The robustness of the model was tested in four ways: (a) by predicting that NF-κB signaling, which requires proteasomal degradation (41), but has no known ciliary link, would be defective in the absence of the proteins of interest described herein; (b) by predicting the direction of the defect in NF-κB signaling; (c) by ameliorating the signaling defects for each tested pathway in vivothrough the chemical upregulation of proteasomal components; and (d) by demonstrating that in the absence of some basal body proteins, the centrosomal proteasome is partially depleted of the various regulatory subunits whose overexpression also has an ameliorating defect in vivo.
Notably, sucrose fraction sedimentation changes were observed in multiple proteasomal subunits in the absence of OFD1 or BBS4, arguing that ciliopathy phenotypes are unlikely to be driven by specific defects in only one subunit, consistent with the observations that the mice haploinsufficient for the subunit RPN10 are phenotypically normal, at least by gross pathology, while homozygous Rpn10−/− mutants are embryonic lethal (42). While not desiring to be bound to any single mechanism of action, an attractive mechanism is one in which basal body proteins regulate the composition of multiple subunits in the proteasome holoenzyme in a context-dependent manner. This is known to occur during cellular stress (43), and it is plausible that ciliary signaling can have a similar effect.
The findings presented herein indicate that targeting the proteasome for compounds that increase its activity can have therapeutic applications for treatment of ciliopathic disorders.
Immunoblotting.
Transfected cells and mouse tissues were lysed in modified RIPA buffer [150 mM sodium chloride, 50 mM Tris-HCl, pH7.4, 1% nonidet P-40, 0.1% sodium deoxycholate, 1 mM EDTA] with 1× proteasome inhibitor (Roche) and centrifuged at 4° C. for 15 minutes. Protein concentration was measured by Lowry assay using the DC Protein Assay Kit (Bio-Rad) on a DU 530 Life Science UV/Vis Spectrophotometer (Beckman Coulter). Total protein in each sample was separated by SDS-PAGE on 4% to 15% Mini-PROTEAN TGX Precast Gel (Bio-Rad) with a Spectra Multicolor Broad Range Protein Ladder (Fermentas) and transferred to an Immun-Blot PVDF Membrane (Bio-Rad). The membrane was blocked with 5% nonfat milk or 5% BSA (Sigma-Aldrich) and probed with the following commercial primary antibodies: anti-GAPDH (ab9484 from Abcam or sc-32233 from Santa Cruz Biotechnology Inc.); anti-GFP (sc-8334; Santa Cruz Biotechnology Inc. or ab13970; Abcam); anti-HSP90 (sc-7947; Santa Cruz Biotechnology Inc.); anti-GLI2 (AF3635; R&D Systems); anti-GLI3 (AF3690; R&D Systems); anti-SUFU (sc-10934; Santa Cruz Biotechnology Inc.); anti-Flag (F7425; Sigma-Aldrich); anti-catenin (sc-7199; Santa Cruz Biotechnology Inc.); anti-α-tubulin (T6199; Sigma-Aldrich); anti-NICD (ab8925; Abcam); anti-hsOFD1 (rabbit polyclonal antisera against human full-length OFD1 NM_003611); anti-mmOFD1 (rabbit polyclonal antisera against a portion of murine OFD1 NM_177429 aa 461-884); anti-BBS4 (AB15009; Millipore); anti-BBS1 (a166613; Abcam); anti-γ-tubulin (T7451; Sigma-Aldrich); anti-20S (NB600-1016; Novus); anti-PA28γ (NBP1-54587; Novus); anti-RPN10 (ab20239; Abcam); anti-RPN13 (H00011037-M01; Novus); anti-RPT2 (AP-107; Boston Biochem); anti-RPT6 (BML-PW9265; Enzo Life Sciences); anti-IκBβ (sc-9248; Santa Cruz Biotechnology Inc.); anti-Myc (m4439; Sigma-Aldrich); and anti-HA (ab16918; Abcam). Densitometric analysis was performed with Image Lab (Bio-Rad), Quantity One (Bio-Rad), or ImageJ 1.44p (NIH) software.
Immunohistochemistry.
Mouse eyes were fixed in 4% PFA, followed by immersion in sucrose (10%, 20%, and 30%) in PBS. With the lens removed, eyecups were embedded in Optimal Cutting Temperature Compound (Sakura) and flash frozen. Cryosections were blocked with 10% FBS in PBS and probed with primary antibodies anti-GFP (ab13970; Abcam) and anti-S-opsin (a gift from Jeremy Nathans, Johns Hopkins University, Baltimore, Md., USA), followed by secondary antibodies Alexa Fluor 488 IgG (Invitrogen) and Alexa Fluor 594 IgG (Invitrogen). Nuclei were stained with DAPI (Roche). Images were captured with a Nikon Eclipse 90i microscope.
Bioinformatic Analyses.
A subset of 271 transcripts were selected a that included: (a) transcripts mutated in human ciliopathies; (b) transcripts that, when mutated in animal models, give rise to ciliary dysfunction; (c) a group of transcripts found in at least to three of the available datasets of ciliary proteins (50); and (d) a subset of transcripts recently shown to be modulators of ciliogenesis and cilium length (51). The publicly available online tool (http://netview.tigem.it) was used to analyze the regulatory interactions among genes from genome-scale measurements of gene expression profiles (microarrays) (32).
ES Cell-Derived In Vitro Neural Differentiation.
The Ofd1-deficient E14Tg2A.4 KOES line was obtained from BayGenomics. Both WT and Ofd1 KOESs were maintained in an undifferentiated state by culture on a monolayer of mitomycin C-inactivated fibroblasts in the presence of leukemia-inhibiting factor (LIF). To induce neural differentiation, previously described protocols were followed (33). Briefly, 48 hours after ES cells were seeded on gelatin-coated plates, they were dissociated and plated on gelatin-coated plates at 1,000 cells/cm2 on day 0 (TO). The culture medium for neuronal differentiation (serum-free KnockOut Serum Replacement-supplemented medium; Invitrogen) contained knockout DMEM supplemented with 15% KSR (Invitrogen), 2 mM L-glutamine, 100 U/ml penicillin-streptomycin, and 0.1 mM β-mercaptoethanol and was replaced daily during the differentiation process.
Cell Culture, DNA Transfection, and Drug Treatment.
HEK-293 or HEK-293-FT cells and human dermal fibroblasts were grown in DMEM (Invitrogen) containing 10% FBS (Invitrogen) and 2 mM L-glutamine (Invitrogen), hTERT-RPE1 cells in DMEM and Ham's F-12 Nutrient 1:1 mixture (DMEM/F-12; Invitrogen) with 10% FBS and 2 mM L-glutamine. FuGene6 Transfection Reagent (Roche) was used for transfection of expression constructs (including the Notch1-NICD expression construct, a gift from Nicholas Gaiano, Johns Hopkins University, Baltimore, Md., USA), shRNA-expressing plasmids, and luciferase reporter plasmids; then cells were cultured for 72 hours. Drug treatment was carried out at a final concentration of 10 μM SFN (Sigma-Aldrich) for 6 to 24 hours, 30 μm N-carbobenzoxyl--leucinyl-
-leucinyl-
-norleucinal (MG132; Calbiochem) for 5 hours, 20 μM lactacystin (EMD Bioscience) for 5 hours, and 50 ng/ml TNF-α (Sigma-Aldrich) for 12 hours.
Immunoprecipitation (IP).
For IP, approximately 1 mg of whole-cell, embryo, or tissue lysate was incubated with anti-OFD1, anti-Flag, or anti-GFP at 4° C. overnight, followed by incubation with protein G-coupled agarose beads (Santa Cruz Biotechnology Inc.) at 4° C. for 1 hour, or directly with anti-Flag M2 beads (A2220 Sigma-Aldrich). The beads were collected and washed with IP buffer (10% glycerol, 50 mM Tris-HCl [pH 7.5], 2.5 mM MgCl2, 1% NP40, and 200 mM NaCl). Proteins conjugated with the beads were then denatured and separated from the beads by boiling at 95° C. to 100° C. for 5 minutes before processing for immunoblotting.
Affinity Purification of Proteasome Complex.
The 26S proteasome complex was purified following a previously described protocol (52) with modifications. Briefly, HEK-293-FT cells expressing stable HTBH-tagged hRPN11 (a gift from Lan Huang, University of California, Irvine, Calif., USA) were transfected with either pSuper control plasmid or pSuperBBS4 to knock down BBS4 expression. Seventy-two hours after transfection, cells were lysed in buffer A (100 mM NaCl, 50 mM Tris-HCl [pH 7.5], 10% glycerol, 2 mM ATP, 1 mM DTT, and 5 mM MgCl2) with 1× proteasome inhibitor (Roche). Lysates were centrifuged at 4° C. for 15 minutes to remove cell debris. To purify proteasomes, an aliquot of the supernatant was incubated with streptavidin beads at 4° C. overnight to precipitate HTBH-RPN11. The beads were then washed with buffer A three times, followed by one washing with TEB buffer (50 mM Tris-HCl, pH 7.5 and 10% glycerol). Finally, the beads were incubated in TEB buffer containing 1% TEV protease at 30° C. for 1 hour, before SDS-PAGE and immunoblotting with anti-RPN10 (ab20239; Abcam).
Immunocytochemistry.
HEK-293 cells cultured on coverslips were fixed in methanol, blocked in normal goat serum (1:10 in PBS containing 5% BSA), and then probed with anti-RPN10 and anti-γ-tubulin, followed by secondary antibodies Alexa Fluor 488 IgG and Alexa Fluor 568 IgG. Finally, nuclei were visualized with Hoechst 33258 (Sigma-Aldrich). Images were captured with a Zeiss LSM 710 confocal microscope and analyzed with ImageJ 1.44p software.
Sucrose Gradient Sedimentation.
HEK-293-FT cells were transfected and treated with nocodazole (10 μg/ml) and cytochalasin B (5 μg/ml) for 1 hour at 72 hours after transfection. To collect cytoplasmic lysates, cells were harvested in lysis buffer (1 mM HEPES [pH 7.3], 0.5% NP-40, 0.5 mM MgCl2, 0.1% β-ME, and 1× protease inhibitor), followed by centrifugation at 2,500 g for 10 minutes. After 10 mM HEPES and 5 U/ml DNase treatment for 30 minutes on ice, 1 ml of cytoplasmic lysates was layered on a discontinued sucrose gradient (70%, 50%, and 40% sucrose in the buffer containing 10 mM PIPES [pH 7.2], 0.1% NP-40, and 0.1% β-ME) and centrifuged for 1 hour at 195,000 g; 2% of lysates were kept before ultracentrifugation and served as an input. After ultracentrifugation, 13 fractions were collected and analyzed by immunoblotting.
Microinjection of Morpholinos, mRNA, and Sulforaphane.
Morpholinos against bbs1 (5′-CACACGTCCATCACTAACCAATAGC-3′; SEQ ID NO: 13), bbs4 (5′-CCGTTCTCATAGCGTCGTCCGCCAT-3′; SEQ ID NO: 14), and ofd1 (5′-ATCTTCTCTACTGCAACACACATAC-3′; SEQ ID NO: 15) were purchased from Gene Tools, LLC. Human RPN10, RPN13, and RPT6 mRNA were in vitro transcribed with a mMESSAGE mMACHINE SP6 Kit (Ambion). SFN was dissolved in DMSO (Sigma-Aldrich) at a stock concentration of 1 M and further diluted in water to 10 mM. The morpholino and mRNA or SFN were mixed, and a volume of 0.5 nl was microinjected.
Whole-Mount RNA In Situ Hybridization.
Zebrafish embryos were fixed overnight in 4% PFA at 4° C. Residual pigment was removed by bleaching with 3% H2O2/0.5% KOH. Whole-mount RNA in situ hybridization was performed with a digoxigenin-labeled anti-her4 RNA probe (a gift from Tohru Ishitani, Kyush University, Fukuoka, Kyushu, Japan) synthesized by in vitro transcription (Roche), followed by immunological detection with Anti-Digoxigenin-AP, Fab Fragments (Roche) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate staining (Roche).
Luciferase Reporter System Assays.
HEK-293-FT cells were seeded in 24-well plates at a density of 104 cells/well. After 24 hours, cells were transfected with expression constructs, short-hairpin plasmids, and a 3X-κB-L reporter (a gift from Tom Gilmore, Boston University, Boston, Mass., USA) for NF-κB signaling. A pRL-SV40 plasmid expressing Renilla luciferase was used as an internal control. Seventy-two hours after transfection, cells were lysed with Passive Lysis Buffer (Promega). The luciferase activity of lysates was measured with the Dual Luciferase Reporter Assay System (Promega) on a FLUOstar Omega microplate reader (BMG LABTECH) and analyzed with MARS Data Analysis Software (BMG LABTECH).
Generation of Ofd1fl/y CAG-Cre-ER™ Mice by Tamoxifen Injections.
Ofd1fl/+ females were crossed with CAG-Cre-ER™ mice, a general deletor Cre line in which Cre-ER is ubiquitously expressed after tamoxifen injection. Pregnant mothers were treated with a single i.p. injection of 100 μg tamoxifen/g of weight at E18.5. Tamoxifen (Sigma-Aldrich) was diluted in 10% ethanol and 90% sesame oil at a final concentration of 10 mg/ml. Quantification of Ofd1 inactivation was assessed by quantitative RT-PCR (qRT-PCR) (data not shown).
Real-Time qRT-PCR Analyses.
Total RNA was isolated with TRIzol (Invitrogen). The cDNA was synthesized from 5 μg of total RNA using SuperScript III (Invitrogen). qRT-PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems) on a 7900HT Fast Real-Time PCR System (Applied Biosystems). The primers Nfkbib forward (5′-TTGGCTACGTCACTGAGGATG-3′; SEQ ID NO: 16) and Nfkbib reverse (5′-GCTCATGCTGATGAATCACAGC-3′; SEQ ID NO: 17) were used to test mRNA levels of Nfkbib, while the primers ofd1 forward (5′-TGGCAGACCACTTACAAAGATG-3′; SEQ ID NO: 18) and ofd1 reverse (AGACTGGATGAGGGGTTAATC-3′; SEQ ID NO: 19) were used to examine the conditional knockout efficiency, and the primers gapdh forward (5′-TCTTCTGGGTGGCAGTGAT-3′; SEQ ID NO: 20) and gapdh reverse (5′-TGCACCACCAACTGCTTAGC-3′; SEQ ID NO: 21) were used as internal controls. Real-time data were collected and analyzed with the Sequence Detection System software package, version 2.3 (Applied Biosystems).
Statistics.
A one-tailed Student's t test was performed to compare the means of two populations. In the bar graphs, data represent the mean±SEM of multiple repeats (n≧3). A χ2 test was performed to compare two populations with several subgroups of different proportions. Statistical significance of differences between samples are indicated by *P<0.05, **P<0.01, and ***P<0.001. A P value less than 0.05 was considered significant.
In a separate approach from the experiments described above, an in vivo genome-wide siRNA screening assay was performed to identify genes whose suppression can rescue the aberrant signaling transduction caused by loss of ciliary function. In this siRNA assay, USP35, a deubiquitinase that acts as a negative regulator of the ubiquitin-proteasome-system (UPS), was identified, as well as 12 other targets. This observed rescue effect seen in cells was then further tested in an animal model for 6 of the 13 identified targets. Specifically, a zebrafish model was used to recapitulate pathogenic features in patients. Depletion of 5 of the 6 targets in the zebrafish model, including USP35, was demonstrated to ameliorate the cerebellar and renal defects in the ciliary deficient animal model.
Specifically, a genome-wide RNAi screening was designed and executed with the to aim of isolating genes whose suppression can rescue hyper Wnt/3-cat signaling caused by loss of BBS4 (a well-known ciliary gene). An RPE (human retinal pigmentosa epicedium) cell line was generated that stably expresses 1) shRNA against BBS4 expression and 2) luciferase reporter possessing 8 TCF-binding sites (
Cells were transfected with Qiagen human whole-genome siRNA library, targeting approximately 22,000 genes in 384-well format. The library can be used as two half libraries, in which four non-related siRNAs (two siRNA in each half library) are designed to suppress the expression of one gene. 72 hours post transfection, cells were stimulated with Wnt3a for another four hours. Finally, cells were harvested and subjected for luciferase and LDH (for cell viability) assays. With the notion that some siRNAs may target to the genes that influence cell viability, leading to the identification of false suppressors, luciferase readout of each experiment was normalized with LDH activity to minimize errors caused by the variation of cell viability.
In this primary screening, 29 genes were identified that can significantly (z<−3; p<0.05) reduce the hyper activation of the Wnt/3-cat signaling. To validate these hits further, Axin2 qPCR was performed in the same cell type used for the primary screening with different source of siRNA (Dharmacon); 13 of 29 genes passed the secondary validation.
These genes are listed in Table 1.
After browsing PubMed and OMIM database, six of the genes from Table 1 were chosen for in vivo assay in a zebrafish model (
Among the genes in Table 1, USP35 was particularly notable; it encodes an ubiquitin peptidase, which acts as a negative regulator in UPS (ubiquitin proteasome system)-mediated protein degradation. This gene target was of particular interest given the recent discovery described herein above of a connection between defective UPS and to ciliopathies, wherein ciliary depletion reduces proteasome degradation leading to the accumulation of signaling effector. Therefore, without desiring to be limited to any one particular mechanism of action, it was hypothesized that inhibition of USP35 can promote proteasome-dependent protein degradation, facilitating the clearance of signaling molecules.
To test this hypothesis, an experiment was designed to test whether suppression of usp38 (the zebrafish ortholog of USP35) can rescue bbs4-depletion induced CE (convergent extension) defects, a phenotype caused by hyper activation of Wnt/3-cat pathway during gastrulation. The resulting data indicate that while depletion of bbs4 caused CE defects in ˜80% embryos, co-injection of a usp38-morpholino (usp38-MO) reduced significantly the incidence of pathology and severity in the embryos (30% in Class I and 5% in Class II;
Next phenotypic readout directly relevant to the clinical findings in ciliopathy patients was used to test the rescue efficiency of usp38 suppression. Identical dosages of MO as previous experiments were injected and 3 dpf (days post of fertilization) zebrafish larvae were collected to examine the morphologic development of the cerebellum. As shown in
In addition, as renal cyst formation is a major common feature in ciliopathy patients (and a key contributor to morbidity and mortality), the renal development was examined in 4 dpf zebrafish larva to evaluate the effect of usp38 suppression. In this model, bbs4 morphants consistently displayed atrophy and deficient convolution in proximal tubules, a phenotype reminiscent of renal immaturity reported in BBS patients and one likely source of cysts. Consistent with the CE and cerebellum studies described herein, the data showed that suppression of usp38 ameliorates the renal defects in the bbs4 morphant (
Taken together, the findings described herein demonstrate the efficiency of usp38 suppression to rescue the ciliopathy related phenotypes in zebrafish model. This indicates attenuation of USP35 activity in patients with ciliopathy disorders to improve disease prognosis.
Similar to USP35, the ZIC1 gene identified in the secondary screen was also tested in the zebrafish embryo model for CE rescue, cerebellum rescue, and renal rescue. In addition, each of the 4 gene targets DTX1, PTMA, DRD5, and ENGASE identified in the secondary screen was also tested in the zebrafish embryo model for CE rescue. The results are shown below in Table 2. The data demonstrate the ability of zic1, ptma, drd5, and engase suppression to rescue the CE phenotype in zebrafish model. This indicates attenuation of these gene product activities in patients with ciliopathy disorders to improve disease prognosis.
Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
One skilled in the art will readily appreciate that the presently described subject matter is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.
This application is a continuation of PCT patent application number PCT/US15/22327 filed Mar. 24, 2015, which claims the benefit of U.S. provisional patent application No. 61/969,397 filed Mar. 24, 2014, the disclosure of which is hereby incorporated by reference in its entirety.
The invention was made with government support under Grant No. R01-HD042601 and Grant No. 1 F32 DK094578-01A1 awarded by the National Institute of Health (NIH). The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61969397 | Mar 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US15/22327 | Mar 2015 | US |
| Child | 15276127 | US |
