Targeting an anchored phosphatase-deacetylase unit restores renal ciliary homeostasis
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Evaluation Summary:
This paper will be of interest to cell biologists interested in understanding processes that regulate the biogenesis of the primary cilium, and to others interested in ciliopathies (ie disorders of the primary cilium). The authors have identified a novel pathway that controls the generation and length of primary cilia and show that its effects are mediated at least in part through its effects on regulating the cytoskeleton. They suggest that this pathway may also have clinical relevance for autosomal dominant polycystic disease, but the data and rationale underlying this part of the study wold need to be strengthened.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Pathophysiological defects in water homeostasis can lead to renal failure. Likewise, common genetic disorders associated with abnormal cytoskeletal dynamics in the kidney collecting ducts and perturbed calcium and cAMP signaling in the ciliary compartment contribute to chronic kidney failure. We show that collecting ducts in mice lacking the A-Kinase anchoring protein AKAP220 exhibit enhanced development of primary cilia. Mechanistic studies reveal that AKAP220-associated protein phosphatase 1 (PP1) mediates this phenotype by promoting changes in the stability of histone deacetylase 6 (HDAC6) with concomitant defects in actin dynamics. This proceeds through a previously unrecognized adaptor function for PP1 as all ciliogenesis and cytoskeletal phenotypes are recapitulated in mIMCD3 knock-in cells expressing a phosphatase-targeting defective AKAP220-ΔPP1 mutant. Pharmacological blocking of local HDAC6 activity alters cilia development and reduces cystogenesis in kidney-on-chip and organoid models. These findings identify the AKAP220-PPI-HDAC6 pathway as a key effector in primary cilia development.
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Author Response:
Reviewer #1:
This study reports the novel and interesting finding that AKAP220 knockout leads to a dramatic increase in primary cilia in renal collecting ducts. AKAP220 is known to sequester PKA, GSK3, the Rho GTPase effector IQGAP-1 and PP1. Previous work from this group demonstrated that AKAP220-/- mice exhibit reduced accumulation of apical actin in the kidney attributable to less GTP-loading of RhoA. Relatedly, AKAP220-/- mice display mild defects in aquaporin 2 trafficking. In this work, Golpalan et al examine the effects of AKAP220 mutation on cilia. They demonstrate increased numbers of primary cilia decorating AKAP220-/- collecting ducts. This phenotype is striking as little is known about negative regulators of cilium biogenesis.
The authors also provide evidence that interaction of AKAP220 with protein …
Author Response:
Reviewer #1:
This study reports the novel and interesting finding that AKAP220 knockout leads to a dramatic increase in primary cilia in renal collecting ducts. AKAP220 is known to sequester PKA, GSK3, the Rho GTPase effector IQGAP-1 and PP1. Previous work from this group demonstrated that AKAP220-/- mice exhibit reduced accumulation of apical actin in the kidney attributable to less GTP-loading of RhoA. Relatedly, AKAP220-/- mice display mild defects in aquaporin 2 trafficking. In this work, Golpalan et al examine the effects of AKAP220 mutation on cilia. They demonstrate increased numbers of primary cilia decorating AKAP220-/- collecting ducts. This phenotype is striking as little is known about negative regulators of cilium biogenesis.
The authors also provide evidence that interaction of AKAP220 with protein phosphatase 1 (PP1) is critical for its function. Through PP1, AKAP220 may regulate HDAC6, which may in turn inhibit tubulin acetylation, which may in turn control cilia stability. Aberrant cilia function is implicated in autosomal dominant polycystic kidney disease. The authors also speculate that AKAP220 and tubulin acetylation may have clinical relevance for autosomal dominant polycystic disease. However, it remains unclear how increased cilia biogenesis may affect cell or tissue physiology. This work is of interest to cell biologists seeking to understand the biogenesis of the primary cilium, and to others interested in ciliopathies (i.e., disorders of the primary cilium).
We thank the reviewer 1 for their insightful comments and concur with their assessment that “it remains unclear how increased cilia biogenesis may affect cell or tissue physiology”. This is clearly a topic for further study within the field that will include ourselves and other laboratories.
Reviewer #2:
The authors show that AKAP220 knockout in kidney collecting ducts leads to a pronounced increase in primary cilia. They go on to demonstrate that this effect holds true in multiple different preparations, before clearly demonstrating that the PP1 anchoring site is critical for the normal role of AKAP220 is limiting primary cilia formation.
Although the key overall finding is well supported, I did not find the specific mechanism concerning a AKAP220-PP1-HDAC6 signaling complex/axis csufficiently onvincing. The authors propose that AKAP220 interacts with HDAC6 via PP1, and that within the complex HDAC6 is stabilised through phosphorylation. The knock on effect is efficient deacetylation. Although this complicated mechanism is consistent with the data, three supporting observations towards this specific mechanism come with caveats: (i) in figure 2C, they show an increase in acetyl tubulin by immunoblotting, but the densitometry seems to be the ratio of acetyl tubulin to GAPDH - would it not be more appropriate to reference to total tubulin?
We are encouraged that this reviewer considers that our “overall findings are well supported”. In response to their comments, we have bolstered our evidence that AKAP220 interacts with HDAC6 via PP1 by including new biochemical and imaging data showing that recruitment of the histone deacetylase is attenuated in kidney cells engineered to express a PP1-binding defective mutant of the anchoring protein. These new data are incorporated into figure 3D and supplemental figures S3D-L.
The mechanism investigated in this paper is concerned with absolute levels of acetylated tubulin. Since the levels of both control proteins (alpha tubulin and GAPDH) and do not change between wildtype and AKAP220KO, therefore we chose to normalize to GAPDH. It is important to note that normalizing to total tubulin does not change the result.
Reviewer #3:
The authors had previously generated a mouse line with inactivation of AKAP220, which encodes an A-kinase anchoring protein, and observed defects in their collecting ducts (CD) leading to defects in trafficking of aquaporin 2. While further characterizing the samples, they observed that CD epithelia had increased numbers and length of their primary cilia compared to CD cells of control mice. While some AKAP proteins have been localized to the primary cilium, AKAP220 was not one of them so the authors pursued a systematic series of experiments to determine how AKAP220 has these effects. Using a combination of CRISPR-manipulated renal epithelial cell lines (IMCD cells), drugs/compounds, 3D and organ-on-a chip cell culture systems they present compelling data that show that AKAP220 anchors a complex of HDAC6 and Protein Phosphatase-1 (PP1) that controls the polymerization of actin and thereby affects cilia formation and elongation. Genetic or pharmacologic manipulations that disrupt AKAP220 or its ability to bind to PP1, inhibit HDAC6, or affect actin stability result in a similar phenotype of enhanced ciliogenesis and ciliary length. Given that polycystic kidney disease has been described as a ciliopathy, with the gene products of the two most common forms of the disease (polycystin-1 and polycystin-2) localized to the cilia, they tested whether inhibiting HDAC6 activity might affect cyst growth using a human iPSC organoid system. They found that organoids lacking polycystin-2 treated with tubacin had smaller cyst size compared to vehicle-treated mutants, leading them to propose manipulation of HDAC6 as a tentative therapeutic strategy for human autosomal dominant polycystic kidney disease and for ciliopathies characterized by defects in ciliogenesis.
Strengths: These findings will be of interest to the ciliary community. They have identified a new factor and its associated partners that appear to regulate ciliogenesis. The studies follow a logical progression and are generally well-done with suitable controls, rigorous quantitation, and a reasonable level of replication (all done at least three times). They have used complementary methods (ie. Genetic manipulation, pharmacologic inhibition) to support their model, sometimes in combination to show that the underlying factor targeted by either genetics or drugs work through the same mechanism.
Weaknesses: The major weakness of the report is in its attempt to be translational. Here, the report has a number of serious theoretical and experimental limitations. On the theoretical level, the rationale behind using an HDAC6 inhibitor is unclear given their data and their model. On the one hand, a prior study had reported that a non-specific inhibitor of HDACs slowed cyst growth in an orthologous mouse model of ADPKD. The current work could suggest that HDAC6 was the actual target in the prior work and that a specific inhibitor for HDAC6 should confer the same benefits. On the other hand, there are compelling reports that show that genetic inhibition of ciliogenesis actually attenuates cystic disease in orthologous mouse models of human ADPKD. The current paradigm is that preserved ciliary activity in the absence of Polycystin-1 or Polycystin-2 promotes cystic growth. This would suggest that any intervention that boosts ciliary function could actually worsen disease. And while the authors never directly comment on the functional properties of the "mutant" cilia that result from deletion of AKAP220 or inhibition of HDAC6, they imply that these "enhanced" cilia are functional by suggesting the use of HDAC6 inhibitors as therapy for ciliopathies that are the result of defective biogenesis. Their prior work also provides indirect support for the notion that the enhanced cilia are functional. AKAP220 knock-out mice are reported to be generally functional, apparently lacking phenotypes commonly associated with defective cilia structure or function. These contradictory observations suggest that one or more of the following conclusions: the "mutant" cilia are in fact poorly functional, the HDAC inhibitors are working through a different mechanism than that which has been proposed, or that the assay as used in this report is not a good read-out of cyst-modulating effects. The last point is particularly relevant for this report. The investigators scored effectiveness of tubacin based on the relative rate of growth of cysts treated with different concentrations of tubacin vs vehicle. In this assay, cyst growth is principally driven by rates of cellular proliferation. Tubacin is an anti-proliferative agent with some toxicity, and while it might be highly selective for HDAC6, these studies cannot distinguish between effects mediated through the AKAP22-HDAC6 pathway versus others. In sum, while tubacin or a similarly-acting drug may or may not be effective for slowing cyst growth, there are multiple reasons to think it isn't through the mechanism the authors propose.
We are encouraged that reviewer 3 considers “our studies follow a logical progression and are generally well-done with suitable controls, rigorous quantitation, and a reasonable level of replication”. In terms of weaknesses, our reading of the reviewer’s detailed passage has identified two specific points that we can address.
Lesions in cilia and polycystins are linked to Autosomal Dominant Polycystic Kidney Disease (Hughes et al., 1995; Mochizuki et al., 1996). Although there is general agreement on this point, the molecular details remain unclear and are inherently paradoxical. For example, loss of morphologically intact cilia favors a less severe cystic phenotype (Ma et al., 2013). In contrast, other investigators report that loss of intact primary cilia results in renal cystogenesis (Kolb and Nauli, 2008; Lin et al., 2003). How primary cilia can be pro-cystogenic in one context yet anti-cystogenic in another context remains an unsolved paradox for the field. We appreciate the need for further clarification on this point as raised by reviewer 3. This conundrum is now noted in the discussion on page 34, line 3.
Searching for a therapeutic approach to restore functional primary cilia is the rationale behind our concluding studies. However, the complexity of genetic models for ADPKD and the above mentioned “cilia paradox” limits our ability to accurately predict how pharmacological agents targeting cilia might affect cellular models of cystogenesis. That being said, we realize that HDAC6 inhibitors have been used by other groups to target cyst size (Cebotaru et al., 2016; Yanda et al., 2017). The reviewer is correct in pointing out that the mechanism by which HDAC6 inhibitors act to inhibit cystogenesis could be less than straightforward given the multitude of functions for HDAC6. We have amended the discussion on page 34, line 5to reflect the reviewer’s valid point.
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Evaluation Summary:
This paper will be of interest to cell biologists interested in understanding processes that regulate the biogenesis of the primary cilium, and to others interested in ciliopathies (ie disorders of the primary cilium). The authors have identified a novel pathway that controls the generation and length of primary cilia and show that its effects are mediated at least in part through its effects on regulating the cytoskeleton. They suggest that this pathway may also have clinical relevance for autosomal dominant polycystic disease, but the data and rationale underlying this part of the study wold need to be strengthened.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers …
Evaluation Summary:
This paper will be of interest to cell biologists interested in understanding processes that regulate the biogenesis of the primary cilium, and to others interested in ciliopathies (ie disorders of the primary cilium). The authors have identified a novel pathway that controls the generation and length of primary cilia and show that its effects are mediated at least in part through its effects on regulating the cytoskeleton. They suggest that this pathway may also have clinical relevance for autosomal dominant polycystic disease, but the data and rationale underlying this part of the study wold need to be strengthened.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This study reports the novel and interesting finding that AKAP220 knockout leads to a dramatic increase in primary cilia in renal collecting ducts. AKAP220 is known to sequester PKA, GSK3, the Rho GTPase effector IQGAP-1 and PP1. Previous work from this group demonstrated that AKAP220-/- mice exhibit reduced accumulation of apical actin in the kidney attributable to less GTP-loading of RhoA. Relatedly, AKAP220-/- mice display mild defects in aquaporin 2 trafficking. In this work, Golpalan et al examine the effects of AKAP220 mutation on cilia. They demonstrate increased numbers of primary cilia decorating AKAP220-/- collecting ducts. This phenotype is striking as little is known about negative regulators of cilium biogenesis.
The authors also provide evidence that interaction of AKAP220 with protein …
Reviewer #1 (Public Review):
This study reports the novel and interesting finding that AKAP220 knockout leads to a dramatic increase in primary cilia in renal collecting ducts. AKAP220 is known to sequester PKA, GSK3, the Rho GTPase effector IQGAP-1 and PP1. Previous work from this group demonstrated that AKAP220-/- mice exhibit reduced accumulation of apical actin in the kidney attributable to less GTP-loading of RhoA. Relatedly, AKAP220-/- mice display mild defects in aquaporin 2 trafficking. In this work, Golpalan et al examine the effects of AKAP220 mutation on cilia. They demonstrate increased numbers of primary cilia decorating AKAP220-/- collecting ducts. This phenotype is striking as little is known about negative regulators of cilium biogenesis.
The authors also provide evidence that interaction of AKAP220 with protein phosphatase 1 (PP1) is critical for its function. Through PP1, AKAP220 may regulate HDAC6, which may in turn inhibit tubulin acetylation, which may in turn control cilia stability. Aberrant cilia function is implicated in autosomal dominant polycystic kidney disease. The authors also speculate that AKAP220 and tubulin acetylation may have clinical relevance for autosomal dominant polycystic disease. However, it remains unclear how increased cilia biogenesis may affect cell or tissue physiology. This work is of interest to cell biologists seeking to understand the biogenesis of the primary cilium, and to others interested in ciliopathies (i.e., disorders of the primary cilium).
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Reviewer #2 (Public Review):
The authors show that AKAP220 knockout in kidney collecting ducts leads to a pronounced increase in primary cilia. They go on to demonstrate that this effect holds true in multiple different preparations, before clearly demonstrating that the PP1 anchoring site is critical for the normal role of AKAP220 is limiting primary cilia formation.
Although the key overall finding is well supported, I did not find the specific mechanism concerning a AKAP220-PP1-HDAC6 signaling complex/axis csufficiently onvincing. The authors propose that AKAP220 interacts with HDAC6 via PP1, and that within the complex HDAC6 is stabilised through phosphorylation. The knock on effect is efficient deacetylation. Although this complicated mechanism is consistent with the data, three supporting observations towards this specific …
Reviewer #2 (Public Review):
The authors show that AKAP220 knockout in kidney collecting ducts leads to a pronounced increase in primary cilia. They go on to demonstrate that this effect holds true in multiple different preparations, before clearly demonstrating that the PP1 anchoring site is critical for the normal role of AKAP220 is limiting primary cilia formation.
Although the key overall finding is well supported, I did not find the specific mechanism concerning a AKAP220-PP1-HDAC6 signaling complex/axis csufficiently onvincing. The authors propose that AKAP220 interacts with HDAC6 via PP1, and that within the complex HDAC6 is stabilised through phosphorylation. The knock on effect is efficient deacetylation. Although this complicated mechanism is consistent with the data, three supporting observations towards this specific mechanism come with caveats: (i) in figure 2C, they show an increase in acetyl tubulin by immunoblotting, but the densitometry seems to be the ratio of acetyl tubulin to GAPDH - would it not be more appropriate to reference to total tubulin? (ii) In Fig. 2O, they propose an interaction between AKAP220-HDAC6 supported by proximity ligation assay data. However, no technical information is provided for the technique, and the control of imaging with HDAC6 + AKAP220deltaPP is not included. No other data (such as co-immunoprecipitations) is provided in support of protein complex formation. (iii) In Figure 3W&X (which is referred back to in the introduction), they propose that because tubacin does not increase the % ciliated cells in an AKAP220deltaPP1 knock-in background, this means that the AKAP220-PP1-HDAC6 axis is key. But there is potentially a ceiling effect at play in this experiment in this experiment since ~ 70 % of cells are ciliated in the AKAP220deltaPP1 knock-in background before the inhibitor is added. The mechanism is plausible but should not be considered concrete in the same way as the central observation that AKAP220 knockout leads to a large increase in cilia.
The study switches tack to focus on F-actin regulation by the AKAP220 complex, and then reveals the potential utility of tubacin to treat renal cystogenesis. Despite reservations about the exact mechanism by which AKAP220 knockout or AKAP220deltaPP1 knock-in drives increase primary cilia formation, the primary finding is interesting and well supported, and should spur on follow-up work to understand the role of this interesting signalling complex in more detail since ciliopathies are an important class of disease.
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Reviewer #3 (Public Review):
The authors had previously generated a mouse line with inactivation of AKAP220, which encodes an A-kinase anchoring protein, and observed defects in their collecting ducts (CD) leading to defects in trafficking of aquaporin 2. While further characterizing the samples, they observed that CD epithelia had increased numbers and length of their primary cilia compared to CD cells of control mice. While some AKAP proteins have been localized to the primary cilium, AKAP220 was not one of them so the authors pursued a systematic series of experiments to determine how AKAP220 has these effects. Using a combination of CRISPR-manipulated renal epithelial cell lines (IMCD cells), drugs/compounds, 3D and organ-on-a chip cell culture systems they present compelling data that show that AKAP220 anchors a complex of HDAC6 …
Reviewer #3 (Public Review):
The authors had previously generated a mouse line with inactivation of AKAP220, which encodes an A-kinase anchoring protein, and observed defects in their collecting ducts (CD) leading to defects in trafficking of aquaporin 2. While further characterizing the samples, they observed that CD epithelia had increased numbers and length of their primary cilia compared to CD cells of control mice. While some AKAP proteins have been localized to the primary cilium, AKAP220 was not one of them so the authors pursued a systematic series of experiments to determine how AKAP220 has these effects. Using a combination of CRISPR-manipulated renal epithelial cell lines (IMCD cells), drugs/compounds, 3D and organ-on-a chip cell culture systems they present compelling data that show that AKAP220 anchors a complex of HDAC6 and Protein Phosphatase-1 (PP1) that controls the polymerization of actin and thereby affects cilia formation and elongation. Genetic or pharmacologic manipulations that disrupt AKAP220 or its ability to bind to PP1, inhibit HDAC6, or affect actin stability result in a similar phenotype of enhanced ciliogenesis and ciliary length. Given that polycystic kidney disease has been described as a ciliopathy, with the gene products of the two most common forms of the disease (polycystin-1 and polycystin-2) localized to the cilia, they tested whether inhibiting HDAC6 activity might affect cyst growth using a human iPSC organoid system. They found that organoids lacking polycystin-2 treated with tubacin had smaller cyst size compared to vehicle-treated mutants, leading them to propose manipulation of HDAC6 as a tentative therapeutic strategy for human autosomal dominant polycystic kidney disease and for ciliopathies characterized by defects in ciliogenesis.
Strengths: These findings will be of interest to the ciliary community. They have identified a new factor and its associated partners that appear to regulate ciliogenesis. The studies follow a logical progression and are generally well-done with suitable controls, rigorous quantitation, and a reasonable level of replication (all done at least three times). They have used complementary methods (ie. Genetic manipulation, pharmacologic inhibition) to support their model, sometimes in combination to show that the underlying factor targeted by either genetics or drugs work through the same mechanism.
Weaknesses: The major weakness of the report is in its attempt to be translational. Here, the report has a number of serious theoretical and experimental limitations. On the theoretical level, the rationale behind using an HDAC6 inhibitor is unclear given their data and their model. On the one hand, a prior study had reported that a non-specific inhibitor of HDACs slowed cyst growth in an orthologous mouse model of ADPKD. The current work could suggest that HDAC6 was the actual target in the prior work and that a specific inhibitor for HDAC6 should confer the same benefits. On the other hand, there are compelling reports that show that genetic inhibition of ciliogenesis actually attenuates cystic disease in orthologous mouse models of human ADPKD. The current paradigm is that preserved ciliary activity in the absence of Polycystin-1 or Polycystin-2 promotes cystic growth. This would suggest that any intervention that boosts ciliary function could actually worsen disease. And while the authors never directly comment on the functional properties of the "mutant" cilia that result from deletion of AKAP220 or inhibition of HDAC6, they imply that these "enhanced" cilia are functional by suggesting the use of HDAC6 inhibitors as therapy for ciliopathies that are the result of defective biogenesis. Their prior work also provides indirect support for the notion that the enhanced cilia are functional. AKAP220 knock-out mice are reported to be generally functional, apparently lacking phenotypes commonly associated with defective cilia structure or function. These contradictory observations suggest that one or more of the following conclusions: the "mutant" cilia are in fact poorly functional, the HDAC inhibitors are working through a different mechanism than that which has been proposed, or that the assay as used in this report is not a good read-out of cyst-modulating effects. The last point is particularly relevant for this report. The investigators scored effectiveness of tubacin based on the relative rate of growth of cysts treated with different concentrations of tubacin vs vehicle. In this assay, cyst growth is principally driven by rates of cellular proliferation. Tubacin is an anti-proliferative agent with some toxicity, and while it might be highly selective for HDAC6, these studies cannot distinguish between effects mediated through the AKAP22-HDAC6 pathway versus others. In sum, while tubacin or a similarly-acting drug may or may not be effective for slowing cyst growth, there are multiple reasons to think it isn't through the mechanism the authors propose.
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