PD-linked LRRK2 G2019S mutation impairs astrocyte morphology and synapse maintenance via ERM hyperphosphorylation
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eLife Assessment
This study identifies astrocyte-intrinsic mechanisms by which the LRRK2 G2019S, a mutation linked to familial Parkinson's disease, disrupts synaptic integrity in the anterior cingulate cortex. The findings are convincing, as they rely on a comprehensive set of in vivo and in vitro genetic, biochemical, proteomic, and electrophysiological approaches. They are important because of their translational value, being validated in both mouse models and post-mortem human samples.
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Abstract
Astrocytes are highly complex cells that mediate critical roles in synapse formation and maintenance by establishing thousands of direct contacts with synapses through their perisynaptic processes. Here, we found that the most common Parkinsonism gene mutation, LRRK2 G2019S, enhances the phosphorylation of the ERM proteins (Ezrin, Radixin, and Moesin), components of the perisynaptic astrocyte processes in a subset of cortical astrocytes. The ERM hyperphosphorylation was accompanied by decreased astrocyte morphological complexity and reduced excitatory synapse density and function. Dampening ERM phosphorylation levels in LRRK2 G2019S mouse astrocytes restored both their morphology and the excitatory synapse density in the anterior cingulate cortex. To determine how LRRK2 mutation impacts Ezrin interactome, we used an in vivo BioID proteomic approach, and we found that astrocytic Ezrin interacts with Atg7, a master regulator of autophagy. The Ezrin/Atg7 interaction is inhibited by Ezrin phosphorylation, thus diminished in LRRK2 G2019S astrocytes. Importantly, the Atg7 function is required to maintain proper astrocyte morphology. Our data provide a molecular pathway through which the LRRK2 G2019S mutation alters astrocyte morphology and synaptic density in a brain-region-specific manner.
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eLife Assessment
This study identifies astrocyte-intrinsic mechanisms by which the LRRK2 G2019S, a mutation linked to familial Parkinson's disease, disrupts synaptic integrity in the anterior cingulate cortex. The findings are convincing, as they rely on a comprehensive set of in vivo and in vitro genetic, biochemical, proteomic, and electrophysiological approaches. They are important because of their translational value, being validated in both mouse models and post-mortem human samples.
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Reviewer #1 (Public review):
Summary:
In this study, the authors aim to uncover how the Parkinson's disease-linked LRRK2 G2019S mutation affects synaptic integrity through astrocyte-intrinsic mechanisms. Specifically, they investigate whether LRRK2-driven ERM hyperphosphorylation disrupts astrocyte morphology and excitatory synapse maintenance, with a focus on regional specificity within the cortex.
Strengths:
(1) Novelty and significance: The work provides important insights into non-neuronal contributions to Parkinson's disease (PD) pathology by highlighting a previously underappreciated role of astrocytic ERM signaling in synapse maintenance. This astrocyte-specific mechanism might help explain early cognitive dysfunctions in PD.
(2) Mechanistic depth: The authors present a detailed molecular pathway where the LRRK2 G2019S mutation …
Reviewer #1 (Public review):
Summary:
In this study, the authors aim to uncover how the Parkinson's disease-linked LRRK2 G2019S mutation affects synaptic integrity through astrocyte-intrinsic mechanisms. Specifically, they investigate whether LRRK2-driven ERM hyperphosphorylation disrupts astrocyte morphology and excitatory synapse maintenance, with a focus on regional specificity within the cortex.
Strengths:
(1) Novelty and significance: The work provides important insights into non-neuronal contributions to Parkinson's disease (PD) pathology by highlighting a previously underappreciated role of astrocytic ERM signaling in synapse maintenance. This astrocyte-specific mechanism might help explain early cognitive dysfunctions in PD.
(2) Mechanistic depth: The authors present a detailed molecular pathway where the LRRK2 G2019S mutation increases ERM phosphorylation, disrupting Ezrin-Atg7 interactions critical for astrocyte morphology.
(3) Robust methodology: The study uses a powerful combination of tools, including AAV-mediated gene delivery, BioID-based interactome mapping, PALE labeling, and patch-clamp electrophysiology to link molecular, morphological, and functional changes.
(4) Physiological relevance: Parallel findings in both mouse models and human post-mortem brains suggest conservation of the observed phenotypes and strengthen the relevance to PD pathogenesis.
Weaknesses:
(1) Causal directionality: While ERM hyperphosphorylation is clearly shown to correlate with morphological and synaptic changes, the specific causal hierarchy-especially between Ezrin-Atg7 interaction loss and synapse alteration, is inferred but not definitively proven. For example, a rescue experiment directly restoring Atg7 function alongside Ezrin manipulation could strengthen this point.
(2) Brain region specificity: Although regional differences between ACC and MOp are well documented, the underlying cause of this differential vulnerability remains speculative. Examining astrocyte heterogeneity within cortical layers or via transcriptomic/proteomic profiling could clarify these regional effects.
(3) Autophagy function: While Atg7 knockdown leads to clear morphological changes, autophagic flux (e.g., LC3-II turnover or p62 accumulation) is not directly assessed. This would strengthen the mechanistic link to autophagy disruption.
(4) GFAP-based astrogliosis interpretation: The conclusion that no astrogliosis occurs in LRRK2 G2019S mice is based solely on GFAP staining. However, GFAP-negative reactive states have been reported. Including additional markers would help validate this interpretation.
(5) Impact on neuronal populations: The authors conclude that changes in inhibitory synapse density in the MOp are not rescued by astrocytic Ezrin manipulation and suggest developmental effects on interneurons. However, this is speculative without neuronal cell-type-specific data. Including interneuron density or synaptic connectivity analysis would make this claim more robust.
(6) Despite these limitations, the authors substantially achieve their stated aims. Their results provide strong support for a model in which astrocytic ERM signaling downstream of LRRK2 contributes to region-specific synaptic changes, particularly in the anterior cingulate cortex. While certain mechanistic links-such as the role of Ezrin-Atg7 interaction in synaptic maintenance-would benefit from further functional validation, the study offers a well-supported framework for understanding astrocyte-intrinsic contributions to synaptic dysfunction in Parkinson's disease.
This work is likely to contribute meaningfully to ongoing research in neurodegeneration, glial biology, and synaptic regulation. The methodological approaches - especially the combination of in vivo models with proteomics and electrophysiology - will be of interest to others studying astrocyte function and neuron-glia interactions. More broadly, the study highlights the importance of astrocyte heterogeneity and regional specialization in shaping neural circuit vulnerability, providing a valuable foundation for future investigations.
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Reviewer #2 (Public review):
Summary:
This is an important study that examines the relationship between a Parkinson's 's-associated mutation in LRRK2 kinase and increased ERM phosphorylation in astrocytes, altered excitatory and inhibitory synapse density and function, and a reduction in astrocyte size. The scope is impressively large and includes human and mouse samples, and employs immunolabeling, whole cell patch clamp recording techniques, molecular manipulation in vivo, and BioID. Experiments have appropriate controls, and the outcomes are mostly convincing. The chief weakness is that the study emphasizes scope over depth, such that it falls short of a unifying model of LRRK2-ERM interactions and leave many outcomes difficult to interpret.
The main idea is that the G2019S Parkinson's mutation in LRRK2 increases its kinase activity …
Reviewer #2 (Public review):
Summary:
This is an important study that examines the relationship between a Parkinson's 's-associated mutation in LRRK2 kinase and increased ERM phosphorylation in astrocytes, altered excitatory and inhibitory synapse density and function, and a reduction in astrocyte size. The scope is impressively large and includes human and mouse samples, and employs immunolabeling, whole cell patch clamp recording techniques, molecular manipulation in vivo, and BioID. Experiments have appropriate controls, and the outcomes are mostly convincing. The chief weakness is that the study emphasizes scope over depth, such that it falls short of a unifying model of LRRK2-ERM interactions and leave many outcomes difficult to interpret.
The main idea is that the G2019S Parkinson's mutation in LRRK2 increases its kinase activity and that this either directly or indirectly increases ERM phosphorylation. This excessive ERM phosphorylation is expected to occur within perisynaptic astrocytic processes, reduce astrocyte complexity, and reduce excitatory synapse density and function in ACC. Overexpression of a dominant negative ezrin (phospho-dead) in astrocytes restores their morphology and excitatory synapse density in ACC. This pathway is well supported if taken on its own. But several datapoints presented do not fit this model. The reasoning driving selectivity to ACC and not M1 is not discussed or pursued (is it relevant that pERM levels appear lower in M1 at P21? Do astrocytes in S1 from G2019S mice also show reduced territories?); the differential effects on excitatory versus inhibitory synapses does not fit the model (or is this effect also expected to lie downstream of astrocytes?). Importantly, the effects of ezrin manipulation in wildtype samples (see below) are not integrated into the model, perhaps because the data run counter to expectation.
Specific Concerns and Questions:
(1) Effects in wildtype mice are not fully incorporated into the model. Overexpressing (OE) WT ezrin appears to reduce pERM levels by about half (Figure 1i vs 4B). OE-phospho-dead ezrin also appears to reduce pERM integrated density compared to control levels (same figures). This is not discussed (see also item 2). OE phospho-dead ezrin decreases synapse density and maybe function compared to OE WT ezrin in wildtype mice (4C, 4F), but it is not clear whether or not these data differ from unmanipulated wildtype sections/slices (Figures 2 and 3) because the data are normalized. These synaptic findings in wildtype should also be joined to the morphology findings in wildtype astrocytes, where OE-phospho-dead ezrin reduces astrocyte territory similar to LRRK2-G2019S. The shared morphological outcome is discussed as a potential defect in ERM phospho/dephospho balance, but it was hard to see if this could be similarly related to changes in synapse density.
(2) Labeling for pERMs shown in wildtype mouse and control human is not convincing, but is convincing in the G2019S samples (e.g., Figure 1/S1, Figure 2) (although concentration in perisynaptic astrocytes is not clear). The data presented seem to better support the idea that the mutation confers a pathological gain of ERM phosphorylation (rather than hyperphosphorylation). If the faint labeling in wildtype and control samples is genuine, one would anticipate that pERM labeling would be different in shControl vs. shLrrk2 astrocytes.
(3) Given the data presented, it would seem that overexpressing the BirA2 ezrin construct, like wildtype ezrin, could impact astrocyte biology. If overexpressing a wildtype ezrin reduces pERM levels, then perhaps the BirA2 construct expression already favors a closed conformation. This is not so much a critique of the approach as a request for clarification and to include, if possible, whether there are reasons to believe or data to support that the BirA2 construct adopts both open and closed conformations.
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Reviewer #3 (Public review):
Summary:
Wang et al. reported a new role of LRRK2-GS mutant in astrocyte morphology and synapse maintenance and a potential mechanism that acts through phosphorylation of ERM, which binds to ATG7. In both human LRRK2-GS patients and LRRK2-GS KI mouse brain cortex, they found increased ERM phosphorylation levels. LRRK2-GS alters excitatory and inhibitory synapse densities and functions in the cortex, which can be restored by p-ERM-dead mutant. They further demonstrated that LRRK2 regulates astrocyte morphological complexity in vivo through ERM phosphorylation. Proteomic and biochemistry approaches found that ATG7 interacts with Ezrin, which is inhibited by Ezrin phosphorylation. This provides a potential mechanism by which LRRK2-GS impairs the astrocyte morphology.
Strengths:
(1) Data in human PD patients …
Reviewer #3 (Public review):
Summary:
Wang et al. reported a new role of LRRK2-GS mutant in astrocyte morphology and synapse maintenance and a potential mechanism that acts through phosphorylation of ERM, which binds to ATG7. In both human LRRK2-GS patients and LRRK2-GS KI mouse brain cortex, they found increased ERM phosphorylation levels. LRRK2-GS alters excitatory and inhibitory synapse densities and functions in the cortex, which can be restored by p-ERM-dead mutant. They further demonstrated that LRRK2 regulates astrocyte morphological complexity in vivo through ERM phosphorylation. Proteomic and biochemistry approaches found that ATG7 interacts with Ezrin, which is inhibited by Ezrin phosphorylation. This provides a potential mechanism by which LRRK2-GS impairs the astrocyte morphology.
Strengths:
(1) Data in human PD patients (Figure 1B, C) is impressive, showing a clear increase of p-ERM in LRRK2-GS samples.
(2) Both LRRK2-GS and siLRRK2 show similar phenotypes, supporting both GOF and LOF decrease astrocyte complexity and size.
(3) Using p-ERM-dead and mimic mutants is elegant. The data is striking that the p-ERM-dead mutant can restore LRRK2-GS-induced excitatory synapse density in the ACC and astrocyte territory volume and complexity, while the p-ERM-mimic mutant can restore the siLRRK2 phenotype.
(4) ATG7 binding to Ezrin provides a potential mechanism. It is compelling that siATG7 shows a similar decrease in astrocyte territory volume and complexity, and siATG7 in LRRK2-GS does not enhance the astrocyte phenotype.
Weaknesses:
(1) The authors claim that p-ERM colocalizes with astrocyte marker ALDH1L1, e.g., Figure 1E, F, G, H, J, K. It is hard to tell from the representative images. Given that this is critical for this paper, it would be appreciated if the authors could improve the images and show clear colocalization. The same concern for Figures S1, 2, 3. Validation of the p-ERM antibody is critical. Figure S4, using λ-PPase to eliminate the phosphorylation signal in general, is very helpful. Additional validation of the p-ERM antibody specific to ERM would be appreciated.
(2) Does the total ERM level change /increase in LRRK2-GS samples? The increased p-ERM levels could be because the total ERM level increases. Then, the follow-up question is whether the total ERM level matters to the astrocyte phenotypes seen in the paper.
(3) WT mice carry WT-LRRK2, which also has kinase activity to phosphorylate ERM. So, what are the effects of overexpression of the p-ERM mutants (dead or mimic) on the excitatory and inhibitory synapse densities and functions in WT mouse samples? In Figure 4, statistics should be done comparing WT+Ezrin O/E vs WT+phosphor-dead Ezrin O/E. From what is shown in the graphs, it looks like phosphor-dead Ezrin worsens the phenotype in WT mice, which is opposite to the GS mice. How to explain? The same question for the graphs in Figure 5.
(4) Rab10 is not a robust substrate for the LRRK2-G2019S mutant, and p-Rab10 is very difficult to detect in mouse brains. The specificity of the pRab10 immunostaining signal in Fig. S8 is not certain.
(5) Would ATG7, Ezrin, and LRRK2 form a complex?
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