N-myc downstream regulated gene 1 (ndrg1) functions as a molecular switch for cellular adaptation to hypoxia
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Evaluation Summary:
The manuscript details the function of the N-Myc downstream regulated gene 1 (NDRG1) during induced hypoxia using the anoxic developing zebrafish as a model system. With some additional support for the central claim of a switch for metabolic suppression, this paper will be of interest to scientists with a focus on kidney development, factors that regulate hypoxic survival, and metabolism in response to stress conditions.
(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
Lack of oxygen (hypoxia and anoxia) is detrimental to cell function and survival and underlies many disease conditions. Hence, metazoans have evolved mechanisms to adapt to low oxygen. One such mechanism, metabolic suppression, decreases the cellular demand for oxygen by downregulating ATP-demanding processes. However, the molecular mechanisms underlying this adaptation are poorly understood. Here, we report on the role of ndrg1a in hypoxia adaptation of the anoxia-tolerant zebrafish embryo. ndrg1a is expressed in the kidney and ionocytes, cell types that use large amounts of ATP to maintain ion homeostasis. ndrg1a mutants are viable and develop normally when raised under normal oxygen. However, their survival and kidney function is reduced relative to WT embryos following exposure to prolonged anoxia. We further demonstrate that Ndrg1a binds to the energy-demanding sodium-potassium ATPase (NKA) pump under anoxia and is required for its degradation, which may preserve ATP in the kidney and ionocytes and contribute to energy homeostasis. Lastly, we show that sodium azide treatment, which increases lactate levels under normoxia, is sufficient to trigger NKA degradation in an Ndrg1a-dependent manner. These findings support a model whereby Ndrg1a is essential for hypoxia adaptation and functions downstream of lactate signaling to induce NKA degradation, a process known to conserve cellular energy.
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Evaluation Summary:
The manuscript details the function of the N-Myc downstream regulated gene 1 (NDRG1) during induced hypoxia using the anoxic developing zebrafish as a model system. With some additional support for the central claim of a switch for metabolic suppression, this paper will be of interest to scientists with a focus on kidney development, factors that regulate hypoxic survival, and metabolism in response to stress conditions.
(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):
The authors used two different genetic approaches of reducing ndrg1a in Zebrafish to investigate its role in metabolic suppression in adaptation to hypoxia. Major strengths include high-quality phenotypic data on organismic survival, striking changes of NKA pattern and abundance in vivo by both hypoxia and ndrg1a genetic manipulations. A major weakness is lack of direct data to characterize "metabolic suppression" and evidence causally linking NKA degradation and metabolic suppression to organismic adaptation and survival against hypoxia. Ndrg family proteins have been previously implicated in many other cellular pathways and functions that are independent of NKA regulation. Thus, while roles of ndrg1a in regulating NKA abundance are novel and clearly shown, the underlying mechanisms and physiological …
Reviewer #1 (Public Review):
The authors used two different genetic approaches of reducing ndrg1a in Zebrafish to investigate its role in metabolic suppression in adaptation to hypoxia. Major strengths include high-quality phenotypic data on organismic survival, striking changes of NKA pattern and abundance in vivo by both hypoxia and ndrg1a genetic manipulations. A major weakness is lack of direct data to characterize "metabolic suppression" and evidence causally linking NKA degradation and metabolic suppression to organismic adaptation and survival against hypoxia. Ndrg family proteins have been previously implicated in many other cellular pathways and functions that are independent of NKA regulation. Thus, while roles of ndrg1a in regulating NKA abundance are novel and clearly shown, the underlying mechanisms and physiological significance remain firmly established.
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Reviewer #2 (Public Review):
The manuscript entitled "N-myc downstream regulated gene 1 (NDRG1) functions as a molecular switch for cellular adaptation to hypoxia" seeks to understand the function of NDGRG1 during induced hypoxia as it relates to the decreased expression of an energy demanding sodium-potassium pump expressed in the pronephric duct and ionocytes. The authors create a novel mutation in the NDRG1 gene that is not associated with lethality and use the mutant to characterize expression of a protein component of the sodium-potassium channel in question. Utilization of zebrafish is a major strength of this manuscript as the question would be more difficult to address in other systems as zebrafish are known for their anoxic survival. The authors also use some very nice techniques that include measuring of kidney clearance, …
Reviewer #2 (Public Review):
The manuscript entitled "N-myc downstream regulated gene 1 (NDRG1) functions as a molecular switch for cellular adaptation to hypoxia" seeks to understand the function of NDGRG1 during induced hypoxia as it relates to the decreased expression of an energy demanding sodium-potassium pump expressed in the pronephric duct and ionocytes. The authors create a novel mutation in the NDRG1 gene that is not associated with lethality and use the mutant to characterize expression of a protein component of the sodium-potassium channel in question. Utilization of zebrafish is a major strength of this manuscript as the question would be more difficult to address in other systems as zebrafish are known for their anoxic survival. The authors also use some very nice techniques that include measuring of kidney clearance, measurements of ATP, lactic acid quantification, and PLA to support their hypothesis. Overall, the data suggest that expression of NDGR1 in the PD and ionocytes is required to initiate the degradation of ATP1A1A, a protein used as a readout for degradation of the sodium potassium pump in question. the authors show colocalization of NDGR1 and ATP1A1 that increases after lactic acid build up and presumably during a switch from aerobic respiration to glycolysis. Additional studies also suggest lysosomal and proteosomal pathways that break down the ATP1A1 protein during hypoxia. Some weaknesses associated with the work include a somewhat small sample size across in situ hybridization/immunohistochemistry (3-9 total animals in replicates) for zebrafish related studies. Some of the graphs and quantification lack significance or at a minimum lack an indication of significance and most of the in situ hybridizations from supplemental and some figures are unclear due to what appears as either a growth delay or different stages. This impacts correct interpretation. Many images are also mounted at different angles preventing correct analysis. Overall, the data support a function for NDGR1 in the PD and during hypoxia, the connection with lactate and the proteosome is less strong albeit very interesting. The conclusions suggesting no role for NDRG1 in kidney function and development from early figures is not substantiated and therefore the conclusions should be toned down. The manuscript requires careful re-examination of stages and better image quality, however, in the event that all embryos can be stage matched through somite counts, the paper will have an impact to closely related fields.
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