Cell autonomous role of leucine-rich repeat kinase in protection of dopaminergic neuron survival

Reviewer #1 (Public Review):

Summary:
This is an important work showing that loss of LRRK function causes late-onset dopaminergic neurodegeneration in a cell-autonomous manner. One of the LRRK members, LRRK2, is of significant translational importance as mutations in LRRK2 cause late-onset autosomal dominant Parkinson's disease (PD). While many in the field assume that LRRK2 mutant causes PD via increased LRRK2 activity (i.e., kinase activity), it is not a settled issue as not all disease-causing mutant LRRK2 exhibits increased activity. Further, while LRRK2 inhibitors are under clinical trials for PD, the consequence of chronic, long-term LRRK2 inhibition is unknown. Thus, studies evaluating the long-term impact of LRRK deficit have important translational implications. Moreover, because LRRK proteins, particularly LRRK2, are known to modulate immune response and intracellular membrane trafficking, the study's results and the reagents will be valuable for others interested in LRRK function.

Strengths:
This report describes a mouse model where LRRK1 and LRRK2 genes are conditionally deleted in dopaminergic neurons. Previously, this group showed that while loss of LRRK2 expression does not cause brain phenotype, loss of both LRRK1 and LRRK2 causes a later onset, progressive degeneration of catecholaminergic neurons, Dopaminergic (DAergic) neurons in substantia niga (SN) and Noradrenergic neurons in Locus Coeruleus (LC). However, because LRRK genes are widely expressed with some peripheral phenotypes, it was unknown if the neurodegeneration in LRRK double Knock Out (DKO) was cell autonomous. To rigorously test this question, the authors have generated a double conditional KO allele where both LRRK1 and LRRK2 genes were targeted to contain loxP sites. In my view, this was beyond what is normally required as most investigators might just combine one KO allele with another floxed allele. The authors provide a rigorous validation showing that the Driver (DAT-Cre) is expressed in the majority of DAergic neurons in SN and that LRRK levels are decreased selectively in the ventral midbrain. Using these mice, the authors show that the number of DA neurons is average at 15 but significantly decreased at 20 months of age. Moreover, the authors show that the number of apoptotic neurons is increased by ~2X in aged SN, demonstrating increased ongoing cell death, as well as an increase in activated microglia. The degeneration is limited to DA neurons as LC neurons are not lost as this population does not express DAT. Overall, the mouse genetics and experimental analysis were performed in a rigorous manner and the results were statistically sound and compelling.

Weakness: I only have a few minor comments. First, in PD and other degenerative conditions, axons and terminals loss occurs prior to cell bodies. It might be beneficial to show the status of DAergic markers in the striatum. Second, previous studies indicate that very little, if any, LRRK1 is expressed in SN DAergic neurons. This also seems to be the case with the Allen Brain Atlas profile. Thus, it is preferable that authors discuss the discrepancy as authors seem to imply significant LRRK1 expression in DA neurons.

Revision: I appreciate the authors revising the manuscript with additional data that have clarified my prior comments. They now show that TH levels in the striatum decrease with SNpc neurons. Further, while there is some disagreement regarding the expression LRRK1 in SNpc, the authors provide a convincing case that LRRK1 function is important in SNpc DA neurons.

Reviewer #2 (Public Review):

Summary: In this manuscript, Shen and collaborators described the generation of conditional double knockout (cDKO) mice lacking LRRK1 and LRRK2 selectively in DAT positive dopaminergic neurons. The Authors asked whether selective deletion of both LRRK isoforms could lead to a Parkinsonian phenotype, as previously reported by the same group in germline double LRRK1 and LRRK2 knockout mice (PMID: 29056298). Indeed, cDKO mice developed a late reduction of TH+ neurons in SNpc that partially correlated with the reduction of NeuN+ cells. This was associated with increased apoptotic cell and microglial cell numbers in SNpc. Unlike the constitutive DKO mice described earlier, however, cDKO mice did not replicate the dramatic increase in the number of autophagic vacuoles. The study supports the authors' hypothesis that loss of function rather than gain of function of LRRK2 leads to Parkinson's Disease.

Strengths: The study described for the first time a model where both the Parkinson's disease-associated gene LRRK2 and its homolog LRRK1 are deleted selectively in dopaminergic neurons, offering a new tool to understand the physiopathological role of LRRK2 and the compensating role of LRRK1 in modulating dopaminergic cell function.

Weaknesses: The model has no construct validity since loss of function mutations of LRRK2 are well tolerated in humans and do not lead to Parkinson's disease. The evidence of a Parkinsonian phenotype in these conditional knockout mice is limited and should be considered preliminary.

Reviewer #3 (Public Review):

Kang, Huang, and colleagues have provided new data to address concerns regarding confirmation of LRRK1 and LRRK2 deletion in their mouse model and the functional impact of the modest loss of TH+ neurons observed in the substantia nigra of their double KO mice. In the revised manuscript, the new data around the characterization of the germline-deleted LRRK1 and LRRK2 mice add confidence that LRRK1 and LRRK2 can be deleted using the genetic approach. They have also added new text to the discussion to try and address some of the comments and questions raised regarding how LRRK1/2 loss may impact cell survival and the implications of this work for PD-linked variants in LRRK2 and therapeutic approaches targeting LRRK2.

The new data provides additional support for the author's claims. I have provided below some suggestions for clarification/additions to the text that can be addressed without additional experiments.

1. The authors added additional text highlighting that more studies are warranted in mice where LRRK1/2 are deleted in other CNS cell types (microglia/astrocytes) to understand cell extrinsic drivers of the autophagy deficits observed in their previous work. It still remains unclear how loss of LRRK1/2 leads to increased apoptosis and gliosis in dopaminergic neurons in a cell-intrinsic manner, and, as suggested in the original review, it would be helpful to add some text to the discussion speculating on potential mechanisms by which this might occur.

2. Revisions have been made to the discussion to clarify their rationale around how variants in LRRK2 associated with PD may be loss-of-function to support the relevance of this mouse model to phenotypes observed in PD. However, as written, the argument that PD-linked variants are loss-of-function is based on the fact that the double KO mice have a mild loss of TH+ neurons while the transgenic mice overexpressing PD-linked LRRK2 variants often do not and that early characterization of kinase activity was done in vitro are relatively weak. Given that the majority of evidence generated by many labs in the field supports a gain-of-function mechanism, the discussion should be further tempered to better highlight the uncertainty around this (rather than strongly arguing for a loss-of-function effect). This could include the mention of increased Rab phosphorylation observed in cellular and animal models and opposing consequences on lysosomal function observed in cellular studies in KO and pathogenic variant expressing cells. Further, a reference to the Whiffen et al. 2020 paper mentioned by another reviewer should be included in the discussion for completeness.

Author Response

The following is the authors’ response to the original reviews.

eLife assessment

This work describes new validated conditional double KO (cDKO) mice for LRRK1 and LRRK2 that will be useful for the field, given that LRRK2 is widely expressed in the brain and periphery, and many divergent phenotypes have been attributed previously to LRRK2 expression. The manuscript presents solid data demonstrating that it is the loss of LRRK1 and LRRK2 expression within the SNpc DA cells that is not well tolerated, as it was previously unclear from past work whether neurodegeneration in the LRRK double Knock Out (DKO) was cell autonomous or the result of loss of LRRK1/LRRK2 expression in other types of cells. Future studies may pursue the biochemical mechanisms underlying the reason for the apoptotic cells noted in this study, as here, the LRRK1/LRRK2 KO mice did not replicate the dramatic increase in the number of autophagic vacuoles previously noted in germline global LRRK1/LRRK2 KO mice.

We thank the editors for handling our manuscript and for the succinct summary that recognizes the significance of our findings and points out interesting directions for future studies. We also thank the reviewers for their helpful comments and positive evaluation of our work. Below, we have provided point-by-point responses to the reviewers’ comments.

Reviewer #1 (Public Review):

Summary:

This is an important work showing that loss of LRRK function causes late-onset dopaminergic neurodegeneration in a cell-autonomous manner. One of the LRRK members, LRRK2, is of significant translational importance as mutations in LRRK2 cause late-onset autosomal dominant Parkinson's disease (PD). While many in the field assume that LRRK2 mutant causes PD via increased LRRK2 activity (i.e., kinase activity), it is not a settled issue as not all disease-causing mutant LRRK2 exhibit increased activity. Further, while LRRK2 inhibitors are under clinical trials for PD, the consequence of chronic, long-term LRRK2 inhibition is unknown. Thus, studies evaluating the long-term impact of LRRK deficit have important translational implications. Moreover, because LRRK proteins, particularly LRRK2, are known to modulate immune response and intracellular membrane trafficking, the study's results and the reagents will be valuable for others interested in LRRK function.

Strengths:

This report describes a mouse model where the LRRK1 and LRRK2 gene is conditionally deleted in dopaminergic neurons. Previously, this group showed that while loss of LRRK2 expression does not cause brain phenotype, loss of both LRRK1 and LRRK2 causes a later onset, progressive degeneration of catecholaminergic neurons and dopaminergic (DAergic) neurons in the substantia nigra (SN), and noradrenergic neurons in the locus coeruleus (LC). However, because LRRK genes are widely expressed with some peripheral phenotypes, it was unknown if the neurodegeneration in the LRRK double knockout (DKO) was cell autonomous. To rigorously test this question, the authors have generated a double conditional (cDKO) allele where both LRRK1 and LRRK2 genes were targeted to contain loxP sites. In my view, this was beyond what is usually required, as most investigators might might combine one KO allele with another floxed allele. The authors provide a rigorous validation showing that the Driver (DAT-Cre) is expressed in most DAergic neurons in the SN and that LRRK levers are decreased selectively in the ventral midbrain. Using these mice, the authors show that the number of DAergic neurons is normal at 15 but significantly decreased at 20 months of age. Moreover, the authors show that the number of apoptotic neurons is increased by ~2X in aged SN, demonstrating increased ongoing cell death, as well as an increase in activated microglia. The degeneration is limited to DAergic neurons as LC neurons are not lost as this population does not express DAT. Overall, the mouse genetics and experimental analysis were performed rigorously, and the results were statistically sound and compelling.

Weaknesses:

I only have a few minor comments. First is that in PD and other degenerative conditions, loss of axons and terminals occurs prior to cell bodies. It might be beneficial to show the status of DAergic markers in the striatum. Second, previous studies indicate that very little, if any, LRRK1 is expressed in SN DAergic neurons. This also the case with the Allen Brain Atlas profile. Thus, authors should discuss the discrepancy as authors seem to imply significant LRRK1 expression in DA neurons.

We appreciate the reviewer’s recognition of the importance of the study as well as our rigorous experimental approaches and compelling results. Our responses to the reviewer's two minor comments are below.

1. DAergic markers in the striatum: We performed TH immunostaining in the striatum and quantified TH+ DA terminals in the striatum of DA neuron-specific LRRK cDKO and littermate control mice at the ages of 15 and 24 months. We found similar levels of TH immunoreactivity in the striatum of LRRK cDKO and littermate control mice at the age of 15 months (p = 0.6565, unpaired Student’s t-test) and significantly reduced levels of TH immunoreactivity in the striatum of LRRK cDKO, compared to control mice at the age of 24 months (~19%, p = 0.0215), suggesting an age-dependent loss of dopaminergic terminals in the striatum of DA neuron-specific LRRK cDKO mice. These results are now included as Figure 5 of the revised manuscript.

2. LRRK1 expression in the SNpc: It is shown in the Mouse brain RNA-seq dataset and the Allen Mouse brain ISH dataset (https://www.proteinatlas.org/ENSG00000154237-LRRK1/brain) that LRRK1 is broadly expressed in the mouse brain and is expressed at modest levels in the midbrain, comparable to the cerebral cortex. Indeed, our Western analysis also showed that levels of LRRK1 detected in the dissected ventral midbrain and the cerebral cortex of control mice are similar (40µg total protein loaded per lane; Figure 2E). Furthermore, we previously demonstrated that deletion of LRRK2 (or LRRK1) alone does not cause age-dependent loss of DA neurons in the SNpc, but deletions of both LRRK1 and LRRK2 result in age-dependent loss of DA neurons in LRRK DKO mice, indicating the functional importance of LRRK1 in the protection of DA neuron survival in the aging mouse brain (Tong et al., PNAS 2010, 107: 9879-9884, Giaime et al., Neuron 2017, 96: 796-807).

Reviewer #2 (Public Review):

Summary:

In this manuscript, Shen and collaborators described the generation of cDKO mice lacking LRRK1 and LRRK2 selectively in DAT-positive DAergic neurons. The Authors asked whether selective deletion of both LRRK isoforms could lead to a Parkinsonian phenotype, as previously reported by the same group in germline double LRRK1 and LRRK2 knockout mice (PMID: 29056298). Indeed, cDKO mice developed a late reduction of TH+ neurons in SNpc that partially correlated with the reduction of NeuN+ cells. This was associated with increased apoptotic cell and microglial cell numbers in SNpc.

Unlike the constitutive DKO mice described earlier, however, cDKO mice did not replicate the dramatic increase in the number of autophagic vacuoles. The study supports the authors' hypothesis that loss of function rather than gain of function of LRRK2 leads to PD.

Strengths:

The study described for the first time a model where both the PD-associated gene LRRK2 and its homolog LRRK1 are deleted selectively in DAergic neurons, offering a new tool to understand the physiopathological role of LRRK2 and the compensating role of LRRK1 in modulating DAergic cell function.

Weaknesses:

The model has no construct validity since loss of function mutations of LRRK2 are well-tolerated in humans and do not lead to PD. The evidence of a Parkinsonian phenotype in these cDKO mice is limited and should be considered preliminary.

We thank the reviewer for commenting on the usefulness of this new PD mouse model.

The reviewer did not include a reference citation for the statement "loss of function mutations of LRRK2 are well-tolerated in humans and do not lead to PD." It is possible that the reviewer was referring to a human population study (Whiffin et al., Nat Med 2020, 26: 869-877), entitled "The effect of LRRK2 lossof-function variants in humans." In this study, the authors analyzed 141,456 individuals sequenced in the Genome Aggregation Database, 49,960 exome-sequenced individuals from the UK Biobank, and more than 4 million participants in the 23andMe genotyped dataset, and they looked for human genetic variants predicted to cause loss-of-function of protein-coding genes (pLoF variants). The reported findings were interesting, and the authors were careful in stating their conclusions. However, this is not a linkage study of large pedigrees carrying a single, clear-cut loss-of-function mutation (e.g. large deletions of most exons and coding sequences). Therefore, the experimental evidence is not compelling enough to conclude whether loss-of-function mutations in LRRK2 cause PD or do not cause PD.

The current report is an unbiased genetic study in an effort to reveal the normal physiological role of LRRK in dopaminergic neurons. It was not intended to produce Parkinsonian phenotypes in LRRK cDKO mice, which would be a biased effort. However, the unequivocal discovery of the cell intrinsic role of LRRK in the protection of DA neurons from age-dependent degeneration and apoptotic cell death should be considered seriously, while we contemplate the disease mechanism and how LRRK2 mutations may cause DA neuron loss and PD.

Reviewer #3 (Public Review):

Kang, Huang, and colleagues investigated the impact of LRRK1 and LRRK2 deletion, specifically in dopaminergic neurons, using a novel cDKO mouse model. They observed a significant reduction in DAergic neurons in the substantia nigra in their conditional LRRK1 and LRRK2 KO mice and a corresponding increase in markers of apoptosis and gliosis. This work set out to address a longstanding question within the field around the role and importance of LRRK1 and LRRK2 in DAergic neurons and suggests that the loss of both proteins triggers some neurodegeneration and glial activation.

The studies included in this work are carefully performed and clearly communicated, but additional studies are needed to strengthen further the authors' claims around the consequences of LRRK2 deletion in DAergic neurons.

1. In Figures 2E and F, the authors assess the protein levels of LRRK1 and LRRK2 in their cDKO mouse model to confirm the deletion of both proteins. They observe a mild loss of LRRK1 and LRRK2 signals in the ventral midbrain compared to wild-type animals. While this is not surprising given other cell types that still express LRRK1 and LRRK2 would be present in their dissected ventral midbrain samples, it does not sufficiently confirm that LRRK1 and LRRK2 are not expressed in DAergic neurons. Additional data is needed to more directly demonstrate that LRRK1 and LRRK2 protein levels are reduced in DAergic neurons, including analysis of LRRK1 and LRRK2 protein levels via immunohistochemistry or FACS-based analysis of TH+ neurons.

We thank the reviewer for highlighting this incredibly important but often overlooked issue. We agree that the data in Figure 2E, F alone would be inadequate to validate DA neuron-specific LRRK cDKO mice.

Cell type-specific conditional knockouts are a mosaic with KO cells mixed with other cell types expressing the gene normally. DA neuron-specific cDKO is particularly challenging, as DA neurons are a subset of cells embedded in the ventral midbrain. Rather than using immunostaining, which relies upon specific, good LRRK1 and LRRK2 antibodies for IHC, or FACS sorting of TH+ neurons followed by Western blotting (few cells, mixed cell populations, etc.), we chose a clean genetic approach by generating germline mutant mice carrying the deleted LRRK1 and LRRK2 alleles in all cells from the floxed LRRK1 and LRRK2 alleles. This approach permits characterization of these deletion mutations in germline mutant mice using molecular approaches that yield unambiguous results.

We crossed CMV-Cre deleter mice with floxed LRRK1 and LRRK2 mice to generate respective germline LRRK1 KO and LRRK2 KO mice, in which all cells carry the LRRK1 or LRRK2 deleted alleles that are identical to those in DA neurons of cDKO mice. We then performed Northern, extensive RTPCR followed by sequencing, and Western analyses to show the absence of the full length LRRK1 and LRRK2 mRNA (Figure 1G, H, Figure 1-figure supplement 8 and 10), and the expected truncation of LRRK1 and LRRK2 mRNA (Figure 1-figure supplement 9 and 11), and the absence of LRRK1 and LRRK2 proteins (Figure 1I). These analyses together demonstrate that in the presence of Cre, either CMV-Cre expressed in all cells or DAT-Cre expressed selectively in DA neurons, the floxed LRRK1 and LRRK2 exons are deleted, resulting in null alleles. We further demonstrated the specificity of DAT-Cremediated recombination (deletion) by crossing DAT-Cre mice with a GFP reporter, showing that 99% TH+ DA neurons in the SNpc are also GFP+ (Figure 2A, B), indicating that DAT-Cre-mediated recombination of the floxed alleles occurs in essentially all TH+ DA neurons in the SNpc.

1. The authors observed a significant but modest effect of LRRK1 and LRRK2 deletion on the number of TH+ neurons in the substantia nigra (12-15% loss at 20-24 months of age). It is unclear whether this extent of neuron loss is functionally relevant. To strengthen the impact of these data, additional studies are warranted to determine whether this translates into any PD-relevant deficits in the mice, including motor deficits or alterations in alpha-synuclein accumulation/aggregation.

Yes, the reduction of DA neurons in the SNpc of cDKO mice at the age of 20-24 months is modest. At 15 months of age, the number of TH+ DA neurons in the SNpc is similar between LRRK cDKO mice (10,000 ± 141) and littermate controls (10,077 ± 310, p > 0.9999). At 20 months of age, the number of DA neurons in the SNpc of LRRK cDKO mice (8,948 ± 273) is significantly reduced (-12.7%), compared to control mice (10,244 ± 220, F1,46 = 16.59, p = 0.0002, two-way ANOVA with Bonferroni’s post hoc multiple comparisons, p = 0.0041). By 24 months of age, the number of DA neurons in the SNpc of LRRK cDKO mice (8,188 ± 452) relative to controls (9,675 ± 232, p = 0.0010) is further reduced (15.4%).

Similar results were obtained by an independent quantification by another investigator, also conducted in a genotype blind manner, using the fractionator and optical dissector method, by which TH+ cells were quantified in 25% areas. These results are included as Figure 3-figure supplement 1 in the revised manuscript. Because of the more limited sampling, the quantification data are more variable, compared to quantification of TH+ cells in all areas of the SNpc, shown in Figure 3. With both methods, we quantified TH+ cells in every 10th sections encompassing the entire SNpc (3D structure), as sampling using every 5th or every 10th sections yielded similar results.

We also performed behavioral analysis of LRRK cDKO mice and littermate controls at the ages of 10 and 25 months using the beam walk test (10 mm and 20 mm beam) and the pole test, which are sensitive to impairment of motor coordination. We found that LRRK cDKO mice at 10 months of age showed significantly more hindlimb errors (p = 0.0005, unpaired two-tailed Student’s t-test) and longer traversal time (p = 0.0075) in the 10mm beam walk test, compared to control mice, though their performance is similar in the 20 mm beam walk (hindlimb slips: p = 0.0733, traversal time: p = 0.9796) and in the pole test. At 22 months of age, the performance of LRRK cDKO mice and littermate controls is more variable and worse, compared to the younger mice, and is not significantly different between the genotypic groups. These results are now included as Figure 9 of the revised manuscript.

1. The authors demonstrate that, unlike in the germline LRRK DKO mice, they do not observe any alterations in electron-dense vacuoles via EM. Given their data showing increased apoptosis and gliosis, it remains unclear how the loss of LRRK proteins leads to DAergic neuronal cell loss. Mechanistic studies would be insightful to understand better potential explanations for how the loss of LRRK1 and LRRK2 may impair cellular survival, and additional text should be added to the discussion to discuss potential hypotheses for how this might occur.

We agree that this phenotypic difference between germline DKO and DA neuron-specific cDKO mice is intriguing, suggesting a non-cell autonomous contribution of LRRK in age-dependent accumulation of autophagic and lysosomal vacuoles in SNpc neurons of germline LRRK DKO mice. We will discuss the phenotypic difference further in the revised manuscript. We are generating microglial specific LRRK cDKO mice to investigate the role of LRRK in microglia and whether microglia contribute in a cell extrinsic manner to the regulation of the autophagy-lysosomal pathway in DA neurons.

1. The authors discuss the potential implications of the neuronal cell loss observed in cDKO mice for LRRK1 and LRRK2 for therapeutic approaches targeting LRRK2 and suggest this argues that LRRK2 variants may exert their effects through a loss-of-protein function. However, all of the data generated in this work focus on a mouse in which both LRRK1 and LRRK2 have been deleted, and it is therefore difficult to make any definitive conclusions about the consequences of specifically targeting LRRK2. The authors note potential redundancy between the two LRRK proteins, and they should soften some of their conclusions in the discussion section around implications for the effects of LRRK2 variants. Human subjects that carry LRRK2 loss-of-function alleles do not have an increased risk for developing PD, which argues against the author's conclusions that LRRK2 variants associated with PD are loss-o-ffunction. Additional text should be included in their discussion to better address these nuances and caution should be used in terms of extrapolating their data to effects observed with PD-linked variants in LRRK2.

We will modify the discussion accordingly in the revised manuscript.