Redox Dyshomeostasis Links Renal and Neuronal Dysfunction in Drosophila Models of Gaucher and Parkinson’s Disease

Reviewer #1 (Public review):

This study investigates the contribution of renal dysfunction to systemic and neuronal decline in Drosophila models of Gaucher disease (Gba1b mutants) and Parkinson's disease (Parkin mutants). While lysosomal and mitochondrial pathways are known drivers in these disorders, the role of kidney-like tissues in disease progression has not been well explored.

The authors use Drosophila melanogaster to model renal dysfunction, focusing on Malpighian tubules (analogous to renal tubules) and nephrocytes (analogous to podocytes). They employ genetic mutants, tissue-specific rescues, imaging of renal architecture, redox probes, functional assays, nephrocyte dextran uptake, and lifespan analyses. They also test genetic antioxidant interventions and pharmacological treatment.

The main findings show that renal pathology is progressive in Gba1b mutants, marked by Malpighian tubule disorganization, stellate cell loss, lipid accumulation, impaired water and ion regulation, and reduced nephrocyte filtration. A central theme is redox dyshomeostasis, reflected in whole-fly GSH reduction, paradoxical mitochondrial versus cytosolic redox shifts, reduced ROS signals, increased lipid peroxidation, and peroxisomal impairment. Antioxidant manipulations (Nrf2, Sod1/2, CatA, and ascorbic acid) consistently worsen outcomes, suggesting a fragile redox balance rather than classical oxidative stress. Parkin mutants also develop renal degeneration, with impaired mitophagy and complete nephrocyte dysfunction by 28 days, but their mechanism diverges from that of Gba1b. Rapamycin treatment rescues several renal phenotypes in Gba1b but not in Parkin, highlighting distinct disease pathways.

The authors propose that renal dysfunction is a central disease-modifying feature of Gaucher and Parkinson's disease models, driven by redox imbalance and differential engagement of lysosomal (Gba1b) vs. mitochondrial (Parkin) mechanisms. They suggest that maintaining renal health and redox balance may represent therapeutic opportunities and biomarkers in neurodegenerative disease. This is a significant manuscript that reframes GD/PD pathology through the lens of renal health. The data are extensive. However, several claims are ahead of the evidence and should be supported with additional experiments.

Major Comments:

(1) The abstract frames progressive renal dysfunction as a "central, disease-modifying feature" in both Gba1b and Parkin models, with systemic consequences including water retention, ionic hypersensitivity, and worsened neuro phenotypes. While the data demonstrates renal degeneration and associated physiological stress, the causal contribution of renal defects versus broader organismal frailty is not fully disentangled. Please consider adding causal experiments (e.g., temporally restricted renal rescue/knockdown) to directly establish kidney-specific contributions.

(2) The manuscript shows multiple redox abnormalities in Gba1b mutants (reduced whole fly GSH, paradoxical mitochondrial reduction with cytosolic oxidation, decreased DHE, increased lipid peroxidation, and reduced peroxisome density/Sod1 mislocalization). These findings support a state of redox imbalance, but the driving mechanism remains broad in the current form. It is unclear if the dominant driver is impaired glutathione handling or peroxisomal antioxidant/β-oxidation deficits or lipid peroxidation-driven toxicity, or reduced metabolic flux/ETC activity. I suggest adding targeted readouts to narrow the mechanism.

(3) The observation that broad antioxidant manipulations (Nrf2 overexpression in tubules, Sod1/Sod2/CatA overexpression, and ascorbic acid supplementation) consistently shorten lifespan or exacerbate phenotypes in Gba1b mutants is striking and supports the idea of redox fragility. However, these interventions are broad. Nrf2 influences proteostasis and metabolism beyond redox regulation, and Sod1/Sod2/CatA may affect multiple cellular compartments. In the absence of dose-response testing or controls for potential off-target effects, the interpretation that these outcomes specifically reflect redox dyshomeostasis feels ahead of the data. I suggest incorporating narrower interpretations (e.g., targeting lipid peroxidation directly) to clarify which redox axis is driving the vulnerability.

(4) This manuscript concludes that nephrocyte dysfunction does not exacerbate brain pathology. This inference currently rests on a limited set of readouts: dextran uptake and hemolymph protein as renal markers, lifespan as a systemic measure, and two brain endpoints (LysoTracker staining and FK2 polyubiquitin accumulation). While these data suggest that nephrocyte loss alone does not amplify lysosomal or ubiquitin stress, they may not fully capture neuronal function and vulnerability. To strengthen this conclusion, the authors could consider adding functional or behavioral assays (e.g., locomotor performance)

(5) The manuscript does a strong job of contrasting Parkin and Gba1b mutants, showing impaired mitophagy in Malpighian tubules, complete nephrocyte dysfunction by day 28, FRUMS clearance defects, and partial rescue with tubule-specific Parkin re-expression. These findings clearly separate mitochondrial quality control defects from the lysosomal axis of Gba1b. However, the mechanistic contrast remains incomplete. Many of the redox and peroxisomal assays are only presented for Gba1b. Including matched readouts across both models (e.g., lipid peroxidation, peroxisome density/function, Grx1-roGFP2 compartmental redox status) would make the comparison more balanced and strengthen the conclusion that these represent distinct pathogenic routes.

(6) Rapamycin treatment is shown to rescue several renal phenotypes in Gba1b mutants (water retention, RSC proliferation, FRUMS clearance, lipid peroxidation) but not in Parkin, and mitophagy is not restored in Gba1b. This provides strong evidence that the two models engage distinct pathogenic pathways. However, the therapeutic interpretation feels somewhat overstated. Human relevance should be framed more cautiously, and the conclusions would be stronger with mechanistic markers of autophagy (e.g., Atg8a, Ref(2)p flux in Malpighian tubules) or with experiments varying dose, timing, and duration (short-course vs chronic rapamycin).

(7) Several systemic readouts used to support renal dysfunction (FRUMS clearance, salt stress survival) could also be influenced by general organismal frailty. To ensure these phenotypes are kidney-intrinsic, it would be helpful to include controls such as tissue-specific genetic rescue in Malpighian tubules or nephrocytes, or timing rescue interventions before overt systemic decline. This would strengthen the causal link between renal impairment and the observed systemic phenotypes.

Reviewer #2 (Public review):

Summary:

In the present study, the authors tested renal function in Gba1b-/- flies and its possible effect on neurodegeneration. They showed that these flies exhibit progressive degeneration of the renal system, loss of water homeostasis, and ionic hypersensitivity. They documented reduced glomerular filtration capacity in their pericardial nephrocytes, together with cellular degeneration in microtubules, redox imbalance, and lipid accumulation. They also compared the Gba1b mutant flies to Parkin mutants and evaluated the effect of treatment with the mTOR inhibitor rapamycin. Restoration of renal structure and function was observed only in the Gba1b mutant flies, leading the authors to conclude that the mutants present different phenotypes due to lysosomal stress in Gba1b mutants versus mitochondrial stress in Parkin mutant flies.

Comments:

(1) The authors claim that: "renal system dysfunction negatively impacts both organismal and neuronal health in Gba1b-/- flies, including autophagic-lysosomal status in the brain." This statement implies that renal impairments drive neurodegeneration. However, there is no direct evidence provided linking renal defects to neurodegeneration in this model. It is worth noting that Gba1b-/- flies are a model for neuronopathic Gaucher disease (GD): they accumulate lipids in their brains and present with neurodegeneration and decreased survival, as shown by Kinghorn et al. (The Journal of Neuroscience, 2016, 36, 11654-11670) and by others, which the authors failed to mention (Davis et al., PLoS Genet. 2016, 12: e1005944; Cabasso et al., J Clin Med. 2019, 8:1420; Kawasaki et al., Gene, 2017, 614:49-55).

(2) The authors tested brain pathology in two experiments:

(a) To determine the consequences of abnormal nephrocyte function on brain health, they measured lysosomal area in the brain of Gba1b-/-, Klf15LOF, or stained for polyubiquitin. Klf15 is expressed in nephrocytes and is required for their differentiation. There was no additive effect on the increased lysosomal volume (Figure 3D) or polyubiquitin accumulation (Figure 3E) seen in Gba1b-/- fly brains, implying that loss of nephrocyte viability itself does not exacerbate brain pathology.

(b) The authors tested the consequences of overexpression of the antioxidant regulator Nrf2 in principal cells of the kidney on neuronal health in Gba1b-/- flies, using the c42-GAL4 driver. They claim that "This intervention led to a significant increase in lysosomal puncta number, as assessed by LysoTrackerTM staining (Figure 5D), and exacerbated protein dyshomeostasis, as indicated by polyubiquitin accumulation and increased levels of the ubiquitin-autophagosome trafficker Ref(2)p/p62 in Gba1b-/- fly brains (Figure 5E). Interestingly, Nrf2 overexpression had no significant effect on lysosomal area or ubiquitin puncta in control brains, demonstrating that the antioxidant response specifically in Gba1b-/- flies negatively impacts disease states in the brain and renal system."
Notably, c42-GAL4 is a leaky driver, expressed in salivary glands, Malpighian tubules, and pericardial cells (Beyenbach et al., Am. J. Cell Physiol. 318: C1107-C1122, 2020). Expression in pericardial cells may affect heart function, which could explain deterioration in brain function.

Taken together, the contribution of renal dysfunction to brain health remains debatable.

Based on the above, I believe the title should be changed to: Redox Dyshomeostasis Links Renal and Neuronal Dysfunction in Drosophila Models of Gaucher disease. Such a title will reflect the results presented in the manuscript.

(3) The authors mention that Gba1b is not expressed in the renal system, which means that no renal phenotype can be attributed directly to any known GD pathology. They suggest that systemic factors such as circulating glycosphingolipids or loss of extracellular vesicle-mediated delivery of GCase may mediate renal toxicity. This raises a question about the validity of this model to test pathology in the fly kidney. According to Flybase, there is expression of Gba1b in renal structures of the fly.

(4) It is worth mentioning that renal defects are not commonly observed in patients with Gaucher disease. Relevant literature: Becker-Cohen et al., A Comprehensive Assessment of Renal Function in Patients With Gaucher Disease, J. Kidney Diseases, 2005, 46:837-844.

(5) In the discussion, the authors state: "Together, these findings establish renal degeneration as a driver of systemic decline in Drosophila models of GD and PD..." and go on to discuss a brain-kidney axis in PD. However, since this study investigates a GD model rather than a PD model, I recommend omitting this paragraph, as the connection to PD is speculative and not supported by the presented data.

(6) The claim: "If confirmed, our findings could inform new biomarker strategies and therapeutic targets for GBA1 mutation carriers and other at-risk groups. Maintaining renal health may represent a modifiable axis of intervention in neurodegenerative disease," extends beyond the scope of the experimental evidence. The authors should consider tempering this statement or providing supporting data.

(7) The conclusion, "we uncover a critical and previously overlooked role for the renal system in GD and PD pathogenesis," is too strong given the data presented. As no mechanistic link between renal dysfunction and neurodegeneration has been established, this claim should be moderated.

(8) The relevance of Parkin mutant flies is questionable, and this section could be removed from the manuscript.

Reviewer #3 (Public review):

Summary:

Hull et al examine Drosophila mutants for the Gaucher's disease locus GBA1/Gba1b, a locus that, when heterozygous, is a risk factor for Parkinson's. Focusing on the Malpighian tubules and their function, they identify a breakdown of cell junctions, loss of haemolymph filtration, sensitivity to ionic imbalance, water retention, and loss of endocytic function in nephrocytes. There is also an imbalance in ROS levels between the cytoplasm and mitochondria, with reduced glutathione levels, rescue of which could not improve longevity. They observe some of the same phenotypes in mutants of Parkin, but treatment by upregulation of autophagy via rapamycin feeding could only rescue the Gba1b mutant and not the Parkin mutant.

Strengths:

The paper uses a range of cellular, genetic, and physiological analyses and manipulations to fully describe the renal dysfunction in the GBa1b animals. The picture developed has depth and detail; the data appears sound and thorough.

Weaknesses:

The paper relies mostly on the biallelic Gba1b mutant, which may reflect dysfunction in Gaucher's patients, though this has yet to be fully explored. The claims for the heterozygous allele and a role in Parkinson's is a little more tenuous, making assumptions that heterozygosity is a similar but milder phenotype than the full loss-of-function.

Author response:

Reviewer #1 (Public review):

Major Comments:

(1) The abstract frames progressive renal dysfunction as a "central, disease-modifying feature" in both Gba1b and Parkin models, with systemic consequences including water retention, ionic hypersensitivity, and worsened neuro phenotypes. While the data demonstrates renal degeneration and associated physiological stress, the causal contribution of renal defects versus broader organismal frailty is not fully disentangled. Please consider adding causal experiments (e.g., temporally restricted renal rescue/knockdown) to directly establish kidney-specific contributions.

We concur that this would help strengthen our conclusions. However, manipulating Gba1b in a tissue-specific manner remains challenging due to its propensity for secretion via extracellular vesicles (ECVs). Leo Pallanck and Marie Davis have elegantly shown that ectopic Gba1b expression in neurons and muscles (tissues with low predicted endogenous expression) is sufficient to rescue major organismal phenotypes. Consistent with this, we have been unable to generate clear tissue-specific phenotypes using Gba1b RNAi.

We will pursue more detailed time-course experiments of the progression of renal pathology, (water weight, renal stem cell proliferation, redox defects, etc.) with the goal of identifying earlier-onset phenotypes that potentially drive dysfunction.

(2) The manuscript shows multiple redox abnormalities in Gba1b mutants (reduced whole fly GSH, paradoxical mitochondrial reduction with cytosolic oxidation, decreased DHE, increased lipid peroxidation, and reduced peroxisome density/Sod1 mislocalization). These findings support a state of redox imbalance, but the driving mechanism remains broad in the current form. It is unclear if the dominant driver is impaired glutathione handling or peroxisomal antioxidant/β-oxidation deficits or lipid peroxidation-driven toxicity, or reduced metabolic flux/ETC activity. I suggest adding targeted readouts to narrow the mechanism.

We agree that we have not yet established a core driver of redox imbalance. Identifying one is likely to be challenging, especially as our RNA-sequencing data from aged Gba1b^⁻/⁻ fly heads (Atilano et al., 2023) indicate that several glutathione S-transferases (GstD2, GstD5, GstD8, and GstD9) are upregulated. We can attempt overexpression of GSTs, which has been elegantly shown by Leo Pallanck to ameliorate pathology in Pink1/Parkin mutant fly brains. However, mechanisms that specifically suppress lipid peroxidation or its associated toxicity, independently of other forms of redox damage, remain poorly understood in Drosophila. Our position is there probably will not be one dominant driver of redox imbalance. Notably, CytB5 overexpression has been shown to reduce lipid peroxidation (Chen et al., 2017), and GstS1 has been reported to conjugate glutathione to the toxic lipid peroxidation product 4-HNE (Singh et al., 2001). Additionally, work from the Bellen lab demonstrated that overexpression of lipases, bmm or lip4, suppresses lipid peroxidation-mediated neurodegeneration (Liu et al., 2015). We will therefore test the effects of over-expressing CytB5, bmm and lip4 in Gba1b^⁻/⁻ flies to help further define the mechanism.

(3) The observation that broad antioxidant manipulations (Nrf2 overexpression in tubules, Sod1/Sod2/CatA overexpression, and ascorbic acid supplementation) consistently shorten lifespan or exacerbate phenotypes in Gba1b mutants is striking and supports the idea of redox fragility. However, these interventions are broad. Nrf2 influences proteostasis and metabolism beyond redox regulation, and Sod1/Sod2/CatA may affect multiple cellular compartments. In the absence of dose-response testing or controls for potential off-target effects, the interpretation that these outcomes specifically reflect redox dyshomeostasis feels ahead of the data. I suggest incorporating narrower interpretations (e.g., targeting lipid peroxidation directly) to clarify which redox axis is driving the vulnerability.

We are in agreement that Drosophila Cnc exhibits functional conservation with both Nrf1 and Nrf2, which have well-established roles in proteostasis and lysosomal biology that may exacerbate pre-existing lysosomal defects in Gba1b mutants. In our manuscript, Nrf2 manipulation forms part of a broader framework of evidence, including dietary antioxidant ascorbic acid and established antioxidant effectors CatA, Sod1, and Sod2. Together, these data indicate that Gba1b mutant flies display a deleterious response to antioxidant treatments or manipulations. To further characterise the redox state, we will quantify lipid peroxidation using Bodipy 581/591 and assess superoxide levels via DHE staining under our redox-altering experimental conditions.

As noted above, we will attempt to modulate lipid peroxidation directly through CytB5 and GstS1 overexpression, acknowledging the caveat that this approach may not fully dissociate lipid peroxidation from other aspects of redox stress. We have also observed detrimental effects of PGC1α on the lifespan of Gba1b^⁻/⁻ flies and will further investigate its impact on redox status in the renal tubules.

(4) This manuscript concludes that nephrocyte dysfunction does not exacerbate brain pathology. This inference currently rests on a limited set of readouts: dextran uptake and hemolymph protein as renal markers, lifespan as a systemic measure, and two brain endpoints (LysoTracker staining and FK2 polyubiquitin accumulation). While these data suggest that nephrocyte loss alone does not amplify lysosomal or ubiquitin stress, they may not fully capture neuronal function and vulnerability. To strengthen this conclusion, the authors could consider adding functional or behavioral assays (e.g., locomotor performance)

We will address this suggestion by performing DAM activity assays and climbing assays in the Klf15; Gba1b^⁻/⁻ double mutants.

(5) The manuscript does a strong job of contrasting Parkin and Gba1b mutants, showing impaired mitophagy in Malpighian tubules, complete nephrocyte dysfunction by day 28, FRUMS clearance defects, and partial rescue with tubule-specific Parkin re-expression. These findings clearly separate mitochondrial quality control defects from the lysosomal axis of Gba1b. However, the mechanistic contrast remains incomplete. Many of the redox and peroxisomal assays are only presented for Gba1b. Including matched readouts across both models (e.g., lipid peroxidation, peroxisome density/function, Grx1-roGFP2 compartmental redox status) would make the comparison more balanced and strengthen the conclusion that these represent distinct pathogenic routes.

We agree that park^⁻/⁻ mutants have been characterised in greater detail than park^⁻/⁻. The primary aim of our study was not to provide an exhaustive characterisation of park¹/¹, but rather to compare key shared and distinct mechanisms underlying renal dysfunction. We have included several relevant readouts for park^⁻/⁻ tubules (e.g., Figure 7D and 8H: mito-Grx1-roGFP2; Figure 8J: lipid peroxidation using BODIPY 581/591). To expand our characterisation of park¹/¹ flies, we will express the cytosolic Grx1 reporter and the peroxisomal marker YFP::Pts.

(6) Rapamycin treatment is shown to rescue several renal phenotypes in Gba1b mutants (water retention, RSC proliferation, FRUMS clearance, lipid peroxidation) but not in Parkin, and mitophagy is not restored in Gba1b. This provides strong evidence that the two models engage distinct pathogenic pathways. However, the therapeutic interpretation feels somewhat overstated. Human relevance should be framed more cautiously, and the conclusions would be stronger with mechanistic markers of autophagy (e.g., Atg8a, Ref(2)p flux in Malpighian tubules) or with experiments varying dose, timing, and duration (short-course vs chronic rapamycin).

We will measure Atg8a, polyubiquitin, and Ref(2)P levels in Gba1b^⁻/⁻ and park^¹/¹ tubules following rapamycin treatment. In our previous study focusing on the gut (Atilano et al., 2023), we showed that rapamycin treatment increased lysosomal area, as assessed using LysoTracker^TM. We will extend this analysis to the renal tubules following rapamycin exposure. Another reviewer requested that we adopt more cautious language regarding the clinical translatability of this work, and we will amend this in Version 2.

(7) Several systemic readouts used to support renal dysfunction (FRUMS clearance, salt stress survival) could also be influenced by general organismal frailty. To ensure these phenotypes are kidney-intrinsic, it would be helpful to include controls such as tissue-specific genetic rescue in Malpighian tubules or nephrocytes, or timing rescue interventions before overt systemic decline. This would strengthen the causal link between renal impairment and the observed systemic phenotypes.

As noted in our response to point 1, we currently lack reliable approaches to manipulate Gba1b in a tissue-specific manner. However, we agree that it is important to distinguish kidney-intrinsic dysfunction from generalised organismal frailty. In the park model, we have already performed renal cell-autonomous rescue: re-expression of Park specifically in Malpighian tubule principal cells (C42-Gal4) throughout adulthood partially normalises water retention, whereas brain-restricted Park expression has no effect on renal phenotypes. Because rescuing Park only in the renal tubules is sufficient to correct a systemic fluid-handling phenotype in otherwise mutant animals, these findings indicate that the systemic defects are driven, at least in part, by renal dysfunction rather than nonspecific organismal frailty.

To strengthen this causal link, we will now extend this same tubule-specific Park rescue (C42-Gal4 and the high-fidelity Malpighian tubule driver CG31272-Gal4) to additional systemic readouts raised by the reviewer. Specifically, we will assay FRUMS clearance and salt stress survival in rescued versus non-rescued park mutants to determine whether renal rescue also mitigates these systemic phenotypes.

Reviewer #2 (Public review):

(1) The authors claim that: "renal system dysfunction negatively impacts both organismal and neuronal health in Gba1b-/- flies, including autophagic-lysosomal status in the brain." This statement implies that renal impairments drive neurodegeneration. However, there is no direct evidence provided linking renal defects to neurodegeneration in this model. It is worth noting that Gba1b-/- flies are a model for neuronopathic Gaucher disease (GD): they accumulate lipids in their brains and present with neurodegeneration and decreased survival, as shown by Kinghorn et al. (The Journal of Neuroscience, 2016, 36, 11654-11670) and by others, which the authors failed to mention (Davis et al., PLoS Genet. 2016, 12: e1005944; Cabasso et al., J Clin Med. 2019, 8:1420; Kawasaki et al., Gene, 2017, 614:49-55).

With the caveats noted in the responses below, we show that driving Nrf2 expression using the renal tubular driver C42 results in decreased survival, more extensive renal defects, and increased brain pathology in Gba1b^⁻/⁻ flies, but not in healthy controls. This suggests that a healthy brain can tolerate renal dysfunction without severe pathological consequences. Our findings therefore indicate that in Gba1b^⁻/⁻ flies, there may be an interaction between renal defects and brain pathology. We do not explicitly claim that renal impairments drive neurodegeneration; rather, we propose that manipulations exacerbating renal dysfunction can have organism-wide effects, ultimately impacting the brain.

The reviewer is correct that our Gba1b^⁻/⁻ fly model represents a neuronopathic GD model with age-related pathology. Indeed, we reproduce the autophagic-lysosomal defects previously reported (Kinghorn et al., 2016) in Figure 5. We agree that the papers cited by the reviewer merit inclusion, and in Version 2 we will incorporate them into the following pre-existing sentence in the Results:

“The gut and brain of Gba1b^⁻/⁻ flies, similar to macrophages in GD patients, are characterised by enlarged lysosomes (Kinghorn et al., 2016; Atilano et al., 2023).”

(2) The authors tested brain pathology in two experiments:

(a) To determine the consequences of abnormal nephrocyte function on brain health, they measured lysosomal area in the brain of Gba1b-/-, Klf15LOF, or stained for polyubiquitin. Klf15 is expressed in nephrocytes and is required for their differentiation. There was no additive effect on the increased lysosomal volume (Figure 3D) or polyubiquitin accumulation (Figure 3E) seen in Gba1b-/- fly brains, implying that loss of nephrocyte viability itself does not exacerbate brain pathology.

(b) The authors tested the consequences of overexpression of the antioxidant regulator Nrf2 in principal cells of the kidney on neuronal health in Gba1b-/- flies, using the c42-GAL4 driver. They claim that "This intervention led to a significant increase in lysosomal puncta number, as assessed by LysoTrackerTM staining (Figure 5D), and exacerbated protein dyshomeostasis, as indicated by polyubiquitin accumulation and increased levels of the ubiquitin-autophagosome trafficker Ref(2)p/p62 in Gba1b-/- fly brains (Figure 5E). Interestingly, Nrf2 overexpression had no significant effect on lysosomal area or ubiquitin puncta in control brains, demonstrating that the antioxidant response specifically in Gba1b-/- flies negatively impacts disease states in the brain and renal system."Notably, c42-GAL4 is a leaky driver, expressed in salivary glands, Malpighian tubules, and pericardial cells (Beyenbach et al., Am. J. Cell Physiol. 318: C1107-C1122, 2020). Expression in pericardial cells may affect heart function, which could explain deterioration in brain function.

Taken together, the contribution of renal dysfunction to brain health remains debatable.

Based on the above, I believe the title should be changed to: Redox Dyshomeostasis Links Renal and Neuronal Dysfunction in Drosophila Models of Gaucher disease. Such a title will reflect the results presented in the manuscript

We agree that C42-Gal4 is a leaky driver; unfortunately, this was true for all commonly used Malpighian tubule drivers available when we began the study. A colleague has recommended CG31272-Gal4 from the Perrimon lab’s recent publication (Xu et al., 2024) as a high-fidelity Malpighian tubule driver. If it proves to maintain principal-cell specificity throughout ageing in our hands, we will repeat key experiments using this driver.

(3) The authors mention that Gba1b is not expressed in the renal system, which means that no renal phenotype can be attributed directly to any known GD pathology. They suggest that systemic factors such as circulating glycosphingolipids or loss of extracellular vesicle-mediated delivery of GCase may mediate renal toxicity. This raises a question about the validity of this model to test pathology in the fly kidney. According to Flybase, there is expression of Gba1b in renal structures of the fly.

Our evidence suggesting that Gba1b is not substantially expressed in renal tissue is based on use of the Gba1b-CRIMIC-Gal4 line, which fails to drive expression of fluorescently tagged proteins in the Malpighian tubules and we have previously shown there is no expression within the nephrocytes with this driver line (Atilano et al., 2023). This does not exclude the possibility that Gba1b functions within the tubules. Notably, Leo Pallanck has provided compelling evidence that Gba1b is present in extracellular vesicles (ECVs) and given the role of the Malpighian tubules in haemolymph filtration, these cells are likely exposed to circulating ECVs. The lysosomal defects observed in Gba1b^⁻/⁻ tubules therefore suggest a potential role for Gba1b in this tissue.

John Vaughan and Thomas Clandinin have developed mCherry- and Lamp1.V5-tagged Gba1b constructs. We intend to express these in tissues shown by the Pallanck lab to release ECVs (e.g., neurons and muscle) and examine whether the protein can be detected in the tubules.

(4) It is worth mentioning that renal defects are not commonly observed in patients with Gaucher disease. Relevant literature: Becker-Cohen et al., A Comprehensive Assessment of Renal Function in Patients With Gaucher Disease, J. Kidney Diseases, 2005, 46:837-844.

We have identified five references indicating that renal involvement, while rare, does occur in association with GD. We agree that this is a valid citation and will include it in the revised introductory sentence:

“However, renal dysfunction remains a rare symptom in GD patients (Smith et al., 1978; Chander et al., 1979; Siegel et al., 1981; Halevi et al., 1993).”

(5) In the discussion, the authors state: "Together, these findings establish renal degeneration as a driver of systemic decline in Drosophila models of GD and PD..." and go on to discuss a brain-kidney axis in PD. However, since this study investigates a GD model rather than a PD model, I recommend omitting this paragraph, as the connection to PD is speculative and not supported by the presented data.

Our position is that Gba1b^⁻/⁻ represents a neuronopathic Gaucher disease model with mechanistic relevance to PD. The severity of GBA1 mutations correlates with the extent of GBA1/GCase loss of function and, consequently, with increased PD risk. Likewise, biallelic park^⁻/⁻ mutants cause a severe and heritable form of PD, and the Drosophila park^⁻/⁻ model is a well-established and widely recognised system that has been instrumental in elucidating how Parkin and Pink1 mutations drive PD pathogenesis.

We therefore see no reason to omit this paragraph. While some aspects are inherently speculative, such discussion is appropriate and valuable when addressing mechanisms underlying a complex and incompletely understood disease, provided interpretations remain measured. At no point do we claim that our work demonstrates a direct brain-renal axis. Rather, our data indicate that renal dysfunction is a disease-modifying feature in these models, aligning with emerging epidemiological evidence linking PD and renal impairment.

(6) The claim: "If confirmed, our findings could inform new biomarker strategies and therapeutic targets for GBA1 mutation carriers and other at-risk groups. Maintaining renal health may represent a modifiable axis of intervention in neurodegenerative disease," extends beyond the scope of the experimental evidence. The authors should consider tempering this statement or providing supporting data.

(7) The conclusion, "we uncover a critical and previously overlooked role for the renal system in GD and PD pathogenesis," is too strong given the data presented. As no mechanistic link between renal dysfunction and neurodegeneration has been established, this claim should be moderated.

We agree that these sections may currently overstate our findings. In Version 2, we will revise them to ensure our claims remain balanced, while retaining the key points that arise from our data and clearly indicating where conclusions require confirmation (“if confirmed”) or additional study (“warrants further investigation”).

“If confirmed, our findings could inform new biomarker strategies and therapeutic targets for patients with GD and PD. Maintaining renal health may represent a modifiable axis of intervention in these diseases.”

“We uncover a notable and previously underappreciated role for the renal system in GD and PD, which now warrants further investigation.”

(8) The relevance of Parkin mutant flies is questionable, and this section could be removed from the manuscript.

We intend to include the data for the Parkin loss-of-function mutants, as these provide essential support for the PD-related findings discussed in our manuscript. To our knowledge, this represents the first demonstration that Parkin mutants display defects in Malpighian tubule function and water homeostasis. We therefore see no reason to remove these findings. Furthermore, as Reviewer 1 specifically requested additional experiments using the Park fly model, we plan to incorporate these analyses in the revised manuscript.

Minor comments:

(1) Figure 1G: The FRUMS assay is not shown for Gba1b-/- flies.

The images in Figure 1G illustrate representative stages of dye clearance. We have quantified the clearance time course for both genotypes. During this process, the tubules of Gba1b^⁻/⁻ flies, similar to controls, sequentially resemble each of the three example images. As the Gba1b^⁻/⁻ tubules appear morphologically identical to controls, differing only in population-level clearance dynamics, we do not feel that including additional example images would provide further informative value.

(2) In panels D and F of Figure 2, survival of control and Gba1b-/- flies in the presence of 4% NaCl is presented. However, longevity is different (up to 10 days in D and ~3 days in F for control). The authors should explain this.

We agree. In our experience, feeding-based stress survival assays show considerable variability between experiments, and we therefore interpret results only within individual experimental replicates. We have observed similar variability in oxidative stress, starvation, and xenobiotic survival assays, which may reflect batch-specific or environmental effects.

(3) In Figure 7F, the representative image does not correspond to the quantification; the percentage of endosome-negative nephrocytes seems to be higher for the control than for the park1/1 flies. Please check this.

The example images are correctly oriented. Typically, an endosome-negative nephrocyte shows no dextran uptake, whereas an endosome-positive nephrocyte displays a ring of puncta around the cell periphery. In park¹/¹ mutants, dysfunctional nephrocytes exhibit diffuse dextran staining throughout the cell, accompanied by diffuse DAPI signal, indicating a complete loss of membrane integrity and likely cell death. We have 63× images from the preparations shown in Figure 7F demonstrating this. In Version 2, we will include apical and medial z-slices of the nephrocytes to illustrate these findings (to be added as supplementary data).

(4) In Figure 7H, the significance between control and park1/1 flies in the FRUMS assay is missing.

We observe significant dye clearance from the haemolymph; however, the difference in complete clearance from the tubules does not reach statistical significance. This may speculatively reflect alterations in specific aspects of tubule function, where absorption and transcellular flux are affected, but subsequent clearance from the tubule lumen remains intact. We do not feel that our current data provide sufficient resolution to draw detailed conclusions about tubule physiology at this level.

Reviewer #3 (Public review):

Weaknesses:

The paper relies mostly on the biallelic Gba1b mutant, which may reflect dysfunction in Gaucher's patients, though this has yet to be fully explored. The claims for the heterozygous allele and a role in Parkinson's is a little more tenuous, making assumptions that heterozygosity is a similar but milder phenotype than the full loss-of-function.

We agree with the reviewer that studying heterozygotes may provide valuable insight into GBA1-associated PD. We will therefore assess whether subtle renal defects are detectable in Gba1b^⁻/⁻ heterozygotes. We clearly state that GBA1 mutations act as a risk factor for PD rather than a Mendelian inherited cause. Consistent with findings from Gba heterozygous mice, Gba1b^⁻/⁻ flies display minimal phenotypes (Kinghorn et al. 2016), and any observable effects are expected to be very mild and age dependent.

(1) Figure 1c, the loss of stellate cells. What age are the MTs shown? Is this progressive or developmental?

These experiments were conducted on flies that were three weeks of age, as were all manipulations unless otherwise stated. We will ensure that this information is clearly indicated in the figure legends in Version 2. We did not observe changes in stellate cell number at three days of age, and this result will be included in the supplementary material in Version 2. Our data therefore suggest that this is a progressive phenotype.

(2) I might have missed this, but for Figure 3, do the mutant flies start with a similar average weight, or are they bloated?

We will perform an age-related time course of water weight in response to Reviewer 1’s comments. For all experiments, fly eggs are age-matched and seeded below saturation density to ensure standardised conditions. Gba1b mutant flies do not exhibit any defects in body size or timing of eclosion.

(3) On 2F, add to the graph that 4% NaCl (or if it is KCL) is present for all conditions, just to make the image self-sufficient to read.

Many thanks for the suggestion. We agree that this will increase clarity and will make this amendment in Version 2 of the manuscript

(4) P13 - rephrase, 'target to either the mitochondria or the cytosol' (as it is phrased, it sounds as though you are doing both at the same time).

We agree and we plan to revise the sentence as follows:

Original:

“To further evaluate the glutathione redox potential (E_GSH) in MTs, we utilised the redox-sensitive green, fluorescent biosensor Grx1-roGFP2, targeted to both the mitochondria and cytosol (Albrecht et al., 2011).”

Revised:

“To further evaluate the glutathione redox potential (E_GSH) in MTs, we utilised the redox-sensitive fluorescent biosensor Grx1-roGFP2, targeted specifically to either the mitochondria or the cytosol using mito- or cyto-tags, respectively (Albrecht et al., 2011).”

(5) In 6F - the staining appears more intense in the Park mutant - perhaps add asterisks or arrowheads to indicate the nephrocytes so that the reader can compare the correct parts of the image?

Reviewer 2 reached the same interpretation. Typically, an endosome-negative nephrocyte shows no dextran uptake, whereas an endosome-positive nephrocyte displays a ring of puncta around the cell periphery. In park¹/¹ mutants, dysfunctional nephrocytes exhibit diffuse dextran staining throughout the cell, accompanied by diffuse DAPI signal, indicative of a complete loss of membrane integrity and likely cell death. We have 63× images from the preparations shown in Figure 7F demonstrating this, and in Version 2 we will include apical and medial z-slices of the nephrocytes to illustrate these findings (to be added as supplementary data).

(6) In the main results text - need some description/explanation of the SOD1 v SOD2 distribution (as it is currently understood) in the cell - SOD2 being predominantly mitochondrial. This helps arguments later on.

Thank you for this suggestion. We plan to amend the text as follows:

“Given that Nrf2 overexpression shortens lifespan in Gba1b^⁻/⁻ flies, we investigated the effects of overexpressing its downstream antioxidant targets, Sod1, Sod2, and CatA, both ubiquitously using the tub-Gal4 driver and with c42-Gal4, which expresses in PCs.”

to:

“Given that Nrf2 overexpression shortens lifespan in Gba1b^⁻/⁻ flies, we investigated the effects of overexpressing its downstream antioxidant targets, Sod1, Sod2, and CatA, both ubiquitously using the tub-Gal4 driver and with c42-Gal4, which expresses in PCs. Sod1 and CatA function primarily in the cytosol and peroxisomes, whereas Sod2 is localised to the mitochondria. Sod1 and Sod2 catalyse the dismutation of superoxide radicals to hydrogen peroxide, while CatA subsequently degrades hydrogen peroxide to water and oxygen.”

(7) Figure 1G, what age are the flies? Same for 3D and E, 4C,D,E, 5B - please check the ages of flies for all of the imaging figures; this information appears to have been missed out.

As stated above, all experiments were conducted on three-week-old flies unless otherwise specified. In Version 2 of the manuscript, we will ensure this information is included consistently in the figure legends to prevent any potential confusion.