Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson’s disease (PD) and Crohn’s disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome. Lysosomes are cellular compartments tasked with breaking down large molecules such as lipids or proteins. They perform an essential role in helping cells dispose of obsolete or harmful components; in fact, defects in lysosome function are associated with a range of health conditions. For instance, many genes associated with an increased risk of developing Parkinson’s disease code for proteins required for lysosomes to work properly, such as the kinase LRRK2.Previous work has shown that this enzyme gets recruited to the surface of damaged lysosomes, where it can modulate the function of another set of molecular actors by modifying them through a chemical process known as phosphorylation. Such activity is increased in harmful versions of LRRK2 linked to Parkinson’s disease. However, the molecular mechanisms which control LRRK2 activity or its recruitment to lysosomes remain unclear.To examine this question, Wang, Bondar et al. first performed a targeted screen to identify proteins that can regulate LRRK2 activity. This revealed that Rab12, one of molecular actors that LRRK2 phosphorylates, can in turn modulate the activity of the enzyme. Further imaging and biochemical experiments then showed that Rab12 is recruited to damaged lysosomes and that this step was in fact necessary for LRRK2 to also relocate to these compartments. The data suggest that this Rab12-driven recruitment process increases the local concentration of LRRK2 near its Rab targets on the membrane of damaged lysosomes, and therefore leads to enhanced LRRK2 activity. Crucially, Wang, Bondar et al. showed that Rab12 also plays a role in the increased LRRK2 activity observed with two Parkinson’s disease-linked mutations , suggesting that increased LRRK2 concentration on lysosomes may be a conserved mechanism that leads to increased LRRK2 activity in disease.Overall, these results highlight a new, Rab12-dependent mechanism that results in enhanced activity at the lysosomal membrane with variants associated with Parkinson’s disease, and for LRRK2 in general when lysosomes are damaged. This knowledge will be helpful to develop therapeutic strategies that target LRRK2, and to better understand how increased LRRK2 activity and lysosomal injury may be linked to Parkinson’s disease. LRRK2 can cause monogenic Parkinson’s disease (PD), and coding and non-coding variants in LRRK2 are associated with increased risk for developing sporadic PD and Crohn’s disease  or VPS35 (D620N). Rab10 phosphorylation was significantly reduced with RAB12 knockdown in LRRK2 R1441G KI and VPS35 D620N KI A549 cells  knock-in, homozygous LRRK2 KO, homozygous RAB12 KO, and homozygous VPS35 D620N (GAT/ATT) knock-in was performed using CRISPR/Cas9. Sequence information for generating targeting gRNA, ssODN donor, and PCR primers are as follows:ice.synthego.com). To isolate monoclonal cell populations, edited cell pools were seeded at 1 cell/well using a single cell printer into 96- or 384-well plates. All wells were imaged every 3 days to ensure expansion from a single-cell clone. Clonal populations were screened and identified using the PCR-Sanger-ICE genotyping strategy described above.CRISPR/Cas9-mediated knockout of LRRK2 or RAB12 and CRISPR/Cas9-mediated knock-in of LRRK2 R1441G or VPS35 D620N in A549 cells was performed by Synthego Corporation . To generate these cells, ribonucleoproteins containing the Cas9 protein and synthetic chemically modified sgRNA produced at Synthego were electroporated into the cells using Synthego’s optimized protocol. Editing efficiency was assessed upon recovery, 48 hr post electroporation. Specifically, genomic DNA was extracted from a portion of the cells, PCR amplified, and sequenced using Sanger sequencing. The resulting chromatograms are processed using Synthego Inference of CRISPR edits software  were transduced with lentivirus carrying the transgene cassette for expression of TMEM192-3x-HA. A synthetic cDNA encoding TMEM192-3x-HA was cloned into pLVX-IRES-hygromycin lentiviral vector containing the CMV promoter. Stably expressing cells were selected using resistance to Hygromycin B  supplied in growth medium at 200 μg/mL for 21 days. Following selection, cells were screened for the stable expression of TMEM192-3x-HA in lysosomes by quantifying the percentage of cells with colocalization of anti-HA and anti-LAMP1 by immunofluorescence, and by monitoring cell lysates for expression TMEM192-3x-HA (~30 kDa) by western blot.For ICC, the following secondary antibodies (Thermo Fisher) were used at 1:1000 dilution: goat anti-mouse Alexa-Fluor 488 (A32723), goat anti-rabbit Alexa-Fluor 568 (A11036).For western blot analysis, the following secondary antibodies  were used at 1:20,000 dilution: IRDyes 800CW donkey anti-rabbit IgG (#926-32213), 680RD donkey anti-mouse IgG (#926-68072).A549 cells were transfected with Dharmacon SMARTpool siRNA targeting 14 Rab GTPases, LRRK2 and non-targeting scramble control , using DharmaFECT 1 . Cells were collected 3 days after transfection for protein or mRNA analysis.The total RNA was extracted from cells using RNeasy Plus Micro Kit . cDNA was synthesized from 1 to 2 μg of RNA using Superscript IV VILO master mix (Thermo Fisher #11756050). The cDNA was diluted threefold and 1 μL of diluted cDNA was used as template. To measure the relative expression levels of mRNAs by RT-qPCR, Taqman Fast Advanced Master Mix (Thermo Fisher #4444557) was used, together with gene-specific primers using TaqMan Assays (Thermo Fisher). GAPDH was used as the housekeeping gene. The PCR was run using QuantStudio 6 Flex Real-Time PCR System, 384-well (Thermo Fisher). Gene expression was analyzed using 2^(delta-delta Ct) method with GAPDH as internal controls.LRRK2, pS935 LRRK2, and pT73-Rab10 MSD assays were previously established . BrieflyCells were lysed in lysis buffer  supplemented with cOmplete tablet , phosSTOP (Roche #04906837001), and Benzonase nuclease . Cell lysates were prepared by incubating with NuPage LDS Sample Buffer  and NuPAGE Sample Reducing Agent  for 5 min at 95°C to denature samples. Lysates were loaded onto NuPAGE 4–12% Bis-Tris gels (Thermo Fisher). Proteins were transferred to nitrocellulose membranes  using Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with Rockland blocking buffer at room temperature for 1 hr , incubated with primary antibody (diluted in Blocking Buffer) overnight at 4°C, and then with secondary antibodies  for 1 hr at room temperature. Odyssey CLx Infrared Imaging System (LI-COR) was used for western blot detection and quantitation.HEK293T cells and A549 cells were cultured in DMEM media (Thermo Fisher #11965-092) containing 1% Pen/Strep and 10% FBS . For LLOMe treatment, LLOMe  was added at 1 mM for 2 hr or 4 hr prior to fixation or lysing cells for downstream analysis. Nocodazole  was added at 25 μΜ for 2 hr prior to fixation or live-cell imaging. Cells were routinely screened to confirm the absence of mycoplasma contamination.Doxycycline-inducible cell lines were generated to stably express WT Rab12 or a phospho-deficient mutant of Rab12 (S106A) in RAB12 KO A549 cells. Briefly, lentiviral constructs were generated by cloning 3XFLAG-RAB12 (or RAB12 S106A) into the pLVX-TetOne-Puro vector. Lentivirus was produced by transfecting the plasmids in HEK293T cells using Lenti-X Packaging Single Shots . The media containing lentivirus were collected from transfected cells and were further concentrated by 50-fold using Lenti-X Concentrator . RAB12 KO A549 cells were infected with lentivirus expressing WT 3XFLAG-RAB12 or 3XFLAG-RAB12 S106A mutant. Cells carrying the lentiviral vectors were selected with puromycin (1 μg/mL). To enable the expression of WT Rab12 or the Rab12 S106A mutant, doxycycline  was added in the cell culture for 3 days.x3 transgene as described previously , harvested by scraping into fresh KPBS and pelleted via centrifugation. Cell pellets were resuspended in KPBS + buffer , and cells were fractionated by passing the suspension through a 21 G needle five times followed by centrifugation at 800 × g for 10 min. Post-nuclear supernatant (PNS) was harvested and incubated with anti-HA magnetic beads  for 15 min with end-over-end rotation. Lysosome-bound beads were washed three times with KPBS + buffer, and samples used for immunoblotting were eluted from beads by heating to 95°C for 10 min in 1× NuPAGE LDS Sample Buffer (Thermo Fisher).Lysosomes were isolated by immunoprecipitation from cells expressing the TMEM192-HAeviously  with theFor analysis of pRab10 levels on isolated lysosomes, one confluent 15 cm plate of cells was used per experimental condition. Cells were treated with 1 mM LLOMe (or vehicle) for 2 hr at 37°C prior to isolation of lysosomes via anti-HA immunoprecipitation as described above. Lyso-IP were performed with 60 µL of anti-HA magnetic bead slurry per condition. Immunoblotting for pRab10 and pRab12 levels was performed in parallel with analysis of total Rab10 and Rab12 levels, as detailed above, using 20% of total immunoprecipitated material per condition. pT73 Rab10, pS106 Rab12, total Rab10, total Rab12, and HA band intensities were quantified from immunoblots using ImageStudio Lite software (LI-COR), and the phospho- and total Rab band intensities were normalized to HA band intensity within each experimental condition. Data were normalized to the median value within each replicate and was then normalized to the mean value of vehicle-treated WT samples across replicates. Calculations for the total fraction of Rab12 present on immunoprecipitated lysosomes were performed by extrapolating the quantitated western blot signal of both the IP and PNS fractions out to 100%, and then calculating the percent of total estimated Rab12 signal captured in the IP divided by the total estimated Rab12 signal present in the PNS sample.For analysis of LRRK2 levels on isolated lysosomes, three confluent 15 cm plates of cells (seeded 24 hr prior to the assay start) were used per experimental condition. Cells were treated with 1 mM LLOMe (or vehicle) for 4 hr at 37°C and then lysosomes were isolated via anti-HA immunoprecipitation as described above. Lyso-IP were performed with 150 µL of anti-HA magnetic bead slurry per condition. For immunoblot detection of endogenous LRRK2, 25% of the total immunoprecipitated material (per condition) was loaded onto a 3–8% Tris-Acetate gel (Thermo Fisher), fully resolved gels were transferred to nitrocellulose membranes, probed overnight at 4°C with a 1/500 dilution of mouse anti-LRRK2 , and imaged using standard immunoblotting protocol detailed above. LRRK2 and HA band intensity was quantified from immunoblots using ImageStudio Lite software (LI-COR), LRRK2 intensity was normalized to HA band intensity within each experimental condition, data was normalized to the median value within each replicate, and then was normalized to the mean value of vehicle-treated WT samples across replicates. Calculations for the total fraction of LRRK2 present on immunoprecipitated lysosomes were performed as for Rab12 (see above).WT, RAB12 KO, and LRRK2 KO A549 cells were seeded in 96-well plates , and then treated with vehicle or LLOMe (1 mM). After 2 hr, cells were fixed with 4% PFA for 15 min, permeabilized and blocked with blocking buffer  for 1 hr at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C. pT73 Rab10 antibody , Rab10 antibody , LAMP1 antibody , and HA antibody  were used in the study. After three washes with PBS/0.05% Triton X-100, secondary fluorescently labeled antibodies were diluted in blocking buffer and incubated for 1 hr at room temperature. DAPI (1:1000) and cell mask deep red  were diluted in PBS/0.05% Triton X-100 and incubated for 10 min. After three washes with PBS/0.05% Triton X-100, the cell plates were imaged on an automated confocal high-content imaging system  using a 63× water immersion objective lens with excitation lasers  and preset emission filters. Channels were separated to avoid fluorescence crosstalk. A custom analysis was developed in the Harmony 4.9 image analysis software (Revvity) to enable image analysis. For analysis of puncta intensity, pT73 Rab10 or total Rab10 spots were defined using ‘Finding Spots’ building blocks, and the sum of corrected spot intensity per cell was used to measure puncta signals. The colocalization between pT73 Rab10 or TMEM192-HA puncta and LAMP1-positive lysosomes were measured with object-based analysis. Briefly, pT73 Rab10 or TMEM192-HA and LAMP1 spots were independently defined using separate ‘Find Spots’ building blocks. Colocalized pT73 Rab10 and LAMP1 spots or TMEM192-HA and LAMP1 spots were identified using the geometric center overlap method within the ‘Select Population’ tool. The average number of colocalized spots were calculated per cell from 16 fields per well  or 20 fields per well  and averaged across the well.For the colocalization analysis of Rab12 and organelle markers, HEK293T cells were transfected with mCherry-Rab12 plasmid (pcDNA3.1 vector) using Lipofectamine LTX with Plus Reagent (Thermo Fisher #15338100), and cells were plated onto poly-lysine-coated 96-well plates . Two days after transfection, cells were treated with vehicle or LLOMe (1 mM), with or without nocodazole (25 μΜ). After 2 hr, cells were fixed and immunostained with a LAMP1 antibody  or GM130 antibody (Abcam ab52649). Cell plates were imaged on an automated confocal high-content imaging system  using a 40× water immersion objective lens. For the colocalization analysis of LRRK2 and organelle markers, HEK293T cells stably expressing eGFP-LRRK2 were used. For the colocalization analysis of LRRK2 and Rab12, HEK293T cells stably expressing eGFP-LRRK2 were transfected with mCherry-Rab12 plasmid. After LLOMe and nocodazole treatment, cell plates were imaged using a 63× water immersion objective lens.For image analysis, we used PCC to assess the colocalization between various pairs of fluorophores: (1) mCherry-Rab12 and 488 LAMP1-positive lysosomes, (2) mCherry-Rab12 and 647 GM130-positive Golgi, (3) eGFP-LRRK2 and 568 LAMP1-positive lysosomes, (4) eGFP-LRRK2 and 647 GM130-positive Golgi, and (5) mCherry-Rab12 and eGFP-LRRK2, as the PCC is a commonly used intensity-based measurement to quantify colocalization between two fluorophores. A custom analysis algorithm built in the Harmony image analysis software  was used to calculate PCC. For Rab12 or LRRK2 singly expressing cells, individual nuclei were identified with DAPI and cell boundaries were defined using either the mCherry or GFP fluorescent channel, respectively. Rab12-positive and LRRK2-positive cells were selected after thresholding for extremely low or high intensities. Lysosomes and Golgi were independently segmented using the ‘Find Spots’ building block in Harmony, and PCC for either Rab12 or LRRK2 was calculated on a per-cell basis within these two compartments. The average PCC score was calculated from ~30 fields per well across three independent biological replicates.For Rab12-LRRK2 colocalization, the number of Rab12-LRRK2 co-expressing cells was much lower than their single-expressing counterparts. Therefore, additional steps were taken to manually identify cells that expressed both mCherry-Rab12 and eGFP-LRRK2 at correct levels. mCherry-Rab12 and eGFP-LRRK2 PCC were calculated on a per-cell basis from ~3 wells across three independent biological replicates.Calculation of the percentage of Rab12 and LRRK2 in lysosomes was performed using the Harmony image analysis software  to determine the percentage of mCherry-Rab12 intensity in lysosomes compared to the whole cell. From Rab12-positive cells, lysosomes were segmented using the LAMP1 channel. mCherry-Rab12 sum intensity from lysosomes was calculated and divided by the total sum mCherry-Rab12 intensity within the entire cell. Values were calculated on a per-cell basis, averaged across ~30 fields per well, and across three independent biological replicates. A similar image analysis process was adapted for calculating the percentage of LRRK2 within lysosomes.WT and LRRK2 KO A549 cells were transfected with mCherry Rab12 plasmid (pcDNA3.1 vector) using Lipofectamine LTX with Plus Reagent (Thermo Fisher #15338100), and cells were plated onto poly-lysine-coated 96-well plates . Two days after transfection, cells were treated with vehicle or LLOMe (1 mM). After 2 hr, cells were fixed and immunostained with LAMP1 antibody . Cell plates were imaged on an automated confocal high-content imaging system  using a 63× water immersion objective lens.For the image analysis, cells were identified through a nuclear stain (DAPI) and lysosomes were segmented with LAMP1 staining using the ‘Find Image Region’ building block in the Harmony 5.1 analysis software (Revvity). The total mCherry-Rab12 levels present in lysosomes were determined by calculating the mean fluorescence intensity of the 568 nm channel within the LAMP1 area. Values were measured from mCherry-Rab12 expressing cells  and averaged across wells (~4–6 wells per condition).2 and 37°C condition. The confocal images were taken every 10 min for 90 min in total.HEK293T cells were transfected with eGFP-LRRK2 and mCherry-Rab12 plasmids (pcDNA3.1 vectors) using Lipofectamine LTX with Plus Reagent (Thermo Fisher #15338100), and cells were plated onto poly-lysine-coated 96-well plates . Two days after transfection, cells were incubated with Hoechst 33342  and CellMask Deep Red Plasma membrane Stain  for 10 min. After replacing the cell culture media containing 1 mM LLOMe, the cell plates were immediately started for live-cell imaging on an automated spinning-disk confocal high-content imaging system  using a 40× water immersion objective lens under 5% COFields of view containing cells co-transfected for eGFP-LRRK2 and mCherry-Rab12 were manually selected from the time lapse dataset across three independent experiments. For each field, channel, and timepoint, the z-stack was converted to a 2D image by maximum intensity projection, then the background intensity was estimated by smoothing the image with a Gaussian filter with a kernel standard deviation of 50 pixels (~14.8 μm) using the ndimage module in scipy v1.9.3 . The bacTo better visualize cells co-expressing low levels of LRRK2 and Rab12, the contrast limits were set to between 0 and 125 AU for LRRK2 and between 0 and 600 AU for Rab12. Cells were included in the segmentation if they (1) appeared morphologically healthy, (2) were visible throughout the time series, and (3) co-expressed LRRK2 and Rab12 at levels above background, but below the maximum contrast limit. Cells were segmented using the brush tool in napari using a brush size of 10 pixels (~3.0 μm). Cells were painted to the edge of the cell border including the nucleus but excluding signal from cell debris and other extracellular sources. Tracking cells across frames was typically possible through a combination of proximity and morphology, but where the assignment was ambiguous, those cells were excluded from further analysis (n=2). The resulting dataset contained 55 segmented cells.For each cell, the non-background subtracted LRRK2 and Rab12 signals were extracted and normalized to between 0.0 and 1.0 by subtracting 200 AU and then dividing by 800 AU. Values above 1.0 or below 0.0 were set to 1.0 or 0.0, respectively. For all pixels under the cell mask, the PCC I was calculated between the normalized LRRK2 and Rab12 signals using the pearsonr function in the scipy.stats module. Cell properties such as area, perimeter, and mean intensity in each channel were extracted for each timepoint using the regionprops function in scikit-image v0.19.3 . Cells wData are shown as mean ± SEM, and all statistical analysis was performed in GraphPad Prism 9. Unpaired (or paired) t-tests were used for statistical analyses of experiments with two treatment groups. For more than two groups, analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison, one-way ANOVA with Sidak’s multiple comparison test, one-way ANOVA with Dunnett’s multiple comparison test, repeated measures one-way ANOVA with Dunnett’s multiple comparison or two-way ANOVA with Sidak’s test, as indicated in figure legends. Comparisons were considered significant where *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.The pT73 Rab10 antibody used in these studies is available from the corresponding author upon reasonable request. This valuable study shows that Rab12 regulates LRRK2 activation via damaged lysosomes. The strength of evidence supporting the claim is compelling. Although key questions about the mechanism remain unanswered, these findings provide a useful template for further research. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Vivek Malhotra as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Rab12 regulates LRRK2 activity by promoting its localization to lysosomes" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. Tone down conclusions throughout as indicated in the reviews.2. Verify the LRRK2 dependence of activation of rab10 phosphorylation.3. Demonstrate that Rab12 does in fact have a more important role than Rab29.4. Clarify the discrepancy in Figure 4 (see comments).5. The data do not show lysosomal accumulation of rab12 precedes LRRK2 (point #1 above) and clarify Fig. 3 as described by Reviewer #3.Reviewer #1 (Recommendations for the authors):1. Fig. 2G. The A549 R1441G cells show lower kinase activity than wild-type cells. Does this clone have less LRRK2? These cells should also be listed under Key Reagents.2. Figure 3E would benefit from nocodazole addition to most clearly demonstrate the true co-localization of Rab12 and LRRK2 on lysosomes (rather than concentration in a very crowded perinuclear area).3. Line 326. "Rab12 and LRRK2 showed a primarily cytosolic localization [at baseline]." Most Rabs are 50% on membranes or bound to GDI in cytosol. The images shown highlight massive concentration in one part of the cell upon LLOME addition and the faint, small-vesicle bound Rab12 would appear very diffuse without actually being non-membrane associated or "cytosolic". Perhaps the word "diffuse" would be more precise for the Rab?4. In the Lyso-IPs, there seems to be a lot of Rab10 on lysosomes yet by microscopy that is never seen. Perhaps the tag is pulling down some Golgi membranes for biosynthetic trafficking of the HA-tag? It might be worth mentioning that this represents a very small fraction of total Rab10 if that is the case. Also, the LAMP is lost in LLOME samples and this should also be mentioned.eLife paper but this is a short report in a competitive area where other complementary stories will bolster this work (Biorxiv doi: https://doi.org/10.1101/2023.02.17.529028).5. Normally mechanism is required for an Reviewer #3 (Recommendations for the authors):Although the overall role of Rab12 in activating LRRK2 is well supported by the data, there are several areas where the study could be improved.1. Although it is likely that the reported relationship between Rab12 and LRRK2 is generalizable to other cell types, this remains to be shown outside of A549 cells. Unapanta et al, bioRxiv, 2022 showed that Rab38 can also play an important role in activating LRRK2 in melanocytes and other cell types might employ other LRRK2 activation mechanisms. Although it is beyond the scope of this study, it will eventually be important to see how Rab12 KO in a mouse model affects LRRK2 activity across a range of tissues (similar to what the Alessi lab did for Rab29).2. It is puzzling that even though LLoMe-induced lysosome damage does not increase LRRK2-R1441G abundance at lysosomes , it still promotes LRRK2-dependent phosphorylation of Rab10 by LRRK2-R1441G . This result suggests that something more than recruitment to lysosomes is required for Rab12-dependent LRRK2 activation. At a minimum, some discussion of these results and the potential impacts of LRRK2 activation mechanisms would be helpful. The claim in lines 395-397 that these pathogenic variants are unable to further respond to additional lysosomal stress is undermined by the discrepancy between 4B vs 4D.3. Based on data in Figure 4A, it is claimed that lysosome damage causes LRRK2 to accumulate at lysosomes. However, in Figure 4D the same treatment fails to yield a similar increase in LRRK2 levels at lysosomes. These inconsistencies undermine confidence in these results and should be explained.4. In multiple figures, treatment with LLoMe results in a decrease in the amount of LAMP1 that is recovered in the TMEM192-HA "lysosome" immunoisolations. This effect was not explained. However, it raises a concern about whether LLoMe is truly triggering LRRK2 recruitment to lysosomes versus the possibility that there is also an effect on TMEM192-HA localization. Is LRRK2 getting recruited to lysosomes? Or is TMEM192-HA moving to a different compartment in the endolysosomal pathway? Some simple experiments that test the colocalization between TMEM192-HA and lysosome proteins +/- LLoMe treatment would help to address this.5. Fig. 3A shows phosphorylated Rab12 at lysosomes but does not show total Rab12 at lysosomes. Showing total Rab12 is required to support claims of regulated recruitment.6. The methods section lacks details for cell line generation and validation. How was Cas9 delivered? How were clones selected? How were mutations in Rab12 assessed?7. Details are lacking on the source and identity of the TMEM192-3xHA plasmid.eLife. Your revised article has been evaluated by Vivek Malhotra (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Rab12 regulates LRRK2 activity by facilitating its localization to lysosomes" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:The main concerns that remained were:1) Title is not appropriate. See below comments from Reviewer #1.2) Some of the data interpretations were not appropriate. All reviewers commented on it.3) Please discuss alternate models, see point 3 from Reviewer #2.Reviewer #1 (Recommendations for the authors):I was really hoping to move this forward; unfortunately, the response to the reviewer comments is not sufficient as follows.1. The authors were asked to modify the title. As it stands, the title implies that Rab12 is on lysosomes which it is normally not, and that LRRK2 is only active on lysosomes which is also not true. The title is really important and either needs to indicate that Rab12 promotes localization to DAMAGED lysosomes or be otherwise modified.2. LRRK2 still produces pRab10 in Rab12 knockout cells. The authors claim on Page 13 that Rab12 is necessary and sufficient for Rab10 phosphorylation but this is not correct. It is clear that LRRK2 still produces pRab10 in cells lacking Rab12 from blots in 2A and 1D. In Figure 2C, the authors show significant variability in Rab10 levels. This highlights the importance of normalizing phosphoRab10 levels to total Rab10 levels. Please normalize data to total Rab10 and not GAPDH or other protein. This is standard in this field.3. The only data on Rab12 being on lysosomes comes from lyso-IPs. In order to understand the magnitude of the process being studied here the authors need to state the fraction of the total Rab12 and total LRRK2 in the LysoIPs of Fig. 3 and 4. This is essential. For example, it is possible that Rab12 increases LRRK2 on lysosomes from 2% of total to 4% of total. The reader needs this information.4. The authors were asked to carry out nocodazole experiments to document true lysosome co-localization. 293T cells are poorly adherent and give poor microscopy images with or without LLOME or nocodazole. Why didn't the authors use their A549 cells as in Fig. 3E? Note that a Golgi marker in these cells would have shown complete co-localization with lysosomes in this scenario-this is why nocodazole is so important to separate membrane compartments. The authors need a clear and well controlled experiment showing that the structures they visualize are lysosomes and not Golgi derived vesicles. This circles back to the title of the paper.Reviewer #2 (Recommendations for the authors):The authors have responded to all of the concerns raised. It is nice to see that LRRK2 is not required for the localization of rab12 to the lysosome. What is very clear is that LLOMe recruits rab12 to the lysosome, induces phosphorylation of rab10 and rab12 is required for phosphorylation of rab10. However, a discrepancy remains between the recruitment of LRRK2 to the lysosome by LLOMe, which is not as apparent for wt LRRK2 in Fig. 4D as in 4A, and the phosphorylation of rab10 induced by LLOMe . So it seems likely, as the authors have now included in the results, that mechanisms other than lysosomal recruitment of LRRK2 contribute to the phosphorylation of rab10. Importantly, they have also shown that LRRK2 is responsible for the induction by LLOMe , so I think this is fine, but they should probably include this important consideration in the discussion as well as the results.Reviewer #3 (Recommendations for the authors):The revised manuscript provides strong evidence that Rab12 can regulate the ability of LRRK2 to phosphorylate Rab10 and that this occurs at least in part by regulating the abundance of LRRK2 at lysosomes. Although key questions about the mechanism remain unanswered, these are still important findings. New data was added to the manuscript that helped to address concerns that were raised during the previous review. Below I have provided some suggestions for clarification to the text that should be addressable without the need for new experiments. However, I also remain concerned about some important results and their interpretation (points 1-).1. There is a discrepancy between the strong decrease in LAMP1 levels in the lysosome IP samples in Figure 3A and that is not seen in the LAMP1 immunofluorescence signal in 3C. This should be acknowledged and/or explained in the text.2. Lines 272-275 imply that previous studies (references 32 and 33) also saw reductions in LAMP1 levels on purified lysosomes following LLoMe treatment. However, I could not find evidence of this in the papers that were cited.3. There is still a discrepancy between Figure 4A-C where LLoMe activates LRRK2 (as measured by phosphor-Rab10) even in the context of VPS35-D620N and LRRK2-R1441G and Figure 4D where LLoMe does not change LRRK2 lysosome abundance in the same cells. The authors touched on this in the response to reviewers but the answer was not definitive. The description and interpretation of these results in the manuscript text is confusing as it is unclear to what extent LRRK2 kinase activity is regulated in response to these stimuli versus LRRK2 localization and proximity to substrates. The data suggests that there might be independent changes to LRRK2 localization and activity. It is not necessary for the authors to solve these puzzles but they should be more clearly presented.4. Line 168: The statement that Rab12 is necessary and sufficient is too strong. Some commentary is required to at minimum acknowledge that this study only examined A549 cells and that the extent that the findings are generalizable to other cell types remains unknown.5. Figure 3A and D and Figure 4A-D should also show the total cell lysates rather than just the IP results.6. Figure 4E and Line 505 over-reach in stating that Rab12 mediates LRRK2 recruitment to lysosomes. Proving this would require evidence that a direct interaction between LRRK2 and Rab12 is necessary for LRRK2 recruitment and activation. Essential revisions:Reviewer #1 (Recommendations for the authors):1. Fig. 2G. The A549 R1441G cells show lower kinase activity than wild-type cells. Does this clone have less LRRK2? These cells should also be listed under Key Reagents.The LRRK2 R1441G KI A549 cells show reduced Rab10 phosphorylation when assessed at the whole cell level. This variant is also associated with reduced LRRK2 levels as assessed by MSD-based analysis in cell lysates as shown in However, analysis of both LRRK2 levels and Rab10 phosphorylation on lysosomes isolated from LRRK2 R1441G KI cells shows an increase in both the lysosomal levels of LRRK2 and pT73 Rab10 with this PD-linked variant. These data demonstrate that the LRRK2 R1441G variant leads to a relative increase in LRRK2 activity at the lysosome.We have updated the Key Reagents list to also include all knock-in and knockout A549 cell models that were employed in this study. We thank the reviewer for pointing this omission out in our initial submission.2. Figure 3E would benefit from nocodazole addition to most clearly demonstrate the true co-localization of Rab12 and LRRK2 on lysosomes (rather than concentration in a very crowded perinuclear area).As suggested by the reviewer, we employed nocodazole treatment to assess whether Rab12 and LRRK2 colocalization is maintained when the clustering of late endosomes/lysosomes in the perinuclear region is disrupted by blocking microtubule assembly. Briefly, HEK293 cells overexpressing mCherry-Rab12 and eGFP-LRRK2 were pre-treated with nocodazole (50 uM) for 2.5 hours and then treated with either vehicle or LLOMe (1mM) for 1.5 hours in the continued presence of nocodazole. The distribution of mCherry-Rab12 and eGFP-LRRK2 was then assessed using high-content confocal imaging. Unfortunately, treatment with both LLOMe and nocodazole caused significant cytotoxicity, rendering us unable to quantify the extent of colocalization of Rab12 and LRRK2 with and without nocodazole treatment following lysosomal membrane permeabilization. We were able to observe a few examples of surviving cells in our studies. In these cells, we found that the apparent colocalization of Rab12 and LRRK2 (overlay in white) following LLOMe treatment was maintained with nocodazole treatment . Using this orthogonal approach, we also observed significant colocalization of pT73 Rab10 with the lysosomal marker LAMP1 following LLOMe treatment that is abolished by RAB12 deletion or LRRK2 deletion. These results provide additional confirmation that LRRK2 promotes Rab10 phosphorylation on lysosomes in a Rab12-dependent manner. As suggested by the reviewer, we have added new text (lines 326-328 of the updated manuscript) to address this point: “While Rab10 has been reported to primarily localize to the Golgi and endosomes, our data show that a proportion of Rab10 is localized to lysosomes basally and in response to lysosomal damage.Dev Cell 2020 PMID: 32916093 and Eriksson et al Cell Death Dis 2020 PMID: 32409651). Our hypothesis is that the loss of LAMP1 following lysosomal membrane permeabilization may arise from the redistribution of LAMPs to the cytosol in small vesicles and/or targeted degradation via lysophagy, as suggested by the studies highlighted. Whereas LAMP1 levels are robustly reduced following LLOMe treatment, we observed a minimal impact on the levels of exogenously-expressed TMEM192-3xHA in isolated lysosomes . As suggested by the reviewer, we have added new text to our updated manuscript that discusses the reduction in LAMP1 observed following LLOMe treatment and potential explanations for this effect (lines 299-304).The reviewer also astutely noted the loss of LAMP1 detected in our isolated lysosomes following LLOMe treatment. This is an effect we have observed across our Lyso-IP experiments. Our data is consistent with previous studies that have also observed a loss of lysosomal transmembrane proteins following LLOMe treatment .We agree with the reviewer that the observations from this study open up many interesting questions regarding the mechanism by which Rab12 senses lysosomal damage and promotes enhanced LRRK2-dependent phosphorylation of Rab10 on lysosomes. We have submitted this as a short report to communicate our findings in a timely fashion given the competitive nature of this field, and we hope to provide additional mechanistic insight regarding this process in future work.Reviewer #3 (Recommendations for the authors):Although the overall role of Rab12 in activating LRRK2 is well supported by the data, there are several areas where the study could be improved.1. Although it is likely that the reported relationship between Rab12 and LRRK2 is generalizable to other cell types, this remains to be shown outside of A549 cells. Unapanta et al, bioRxiv, 2022 showed that Rab38 can also play an important role in activating LRRK2 in melanocytes and other cell types might employ other LRRK2 activation mechanisms. Although it is beyond the scope of this study, it will eventually be important to see how Rab12 KO in a mouse model affects LRRK2 activity across a range of tissues (similar to what the Alessi lab did for Rab29).in vivo by analyzing LRRK2-dependent Rab10 phosphorylation across different tissues in Rab12 KO mice . The authors observed reductions in phospho-Rab10 levels in Rab12 KO mouse lung with trends toward reduction in the large intestine and kidney. This study supports the relevance of Rab12 as a regulator of LRRK2 activity beyond the A549 cell model used in the present work. The authors could not assess the role for Rab12 in regulating LRRK2 activity in the brain given challenges in detecting pT73 Rab10 in brain tissue, and additional studies in human iPSC-derived CNS cells are warranted to better assess the consequences of Rab12 deletion in the brain. These additional studies will undoubtedly be a focus of future work by us (and others) but are beyond scope for the present manuscript.The reviewer raises a great question regarding whether Rab12 regulates LRRK2 activity in additional cell types beyond the A549 cell model used in the present work. In a recent preprint that also identified Rab12 as a key regulator of LRRK2 activation, the authors characterize the impact of loss of Rab12 2. It is puzzling that even though LLoMe-induced lysosome damage does not increase LRRK2-R1441G abundance at lysosomes , it still promotes LRRK2-dependent phosphorylation of Rab10 by LRRK2-R1441G . This result suggests that something more than recruitment to lysosomes is required for Rab12-dependent LRRK2 activation. At a minimum, some discussion of these results and the potential impacts of LRRK2 activation mechanisms would be helpful. The claim in lines 395-397 that these pathogenic variants are unable to further respond to additional lysosomal stress is undermined by the discrepancy between 4B vs 4D.The reviewer raises an important point regarding the potential mechanisms by which the LRRK2 R1441G variant leads to increased Rab10 phosphorylation at the lysosome and whether additional factors beyond LRRK2 localization may contribute to this effect. We focused on detecting effects on the localization of endogenously-expressed LRRK2 to avoid any potential artifacts caused by LRRK2 overexpression. Because of this, the amount of LRRK2 detected on isolated lysosomes is low, and technical variability in lysosomal isolations across independent experiments can complicate our ability to quantify smaller effects on LRRK2 levels on lysosomes. To add confidence in the effect of lysosomal damage on LRRK2 levels in LRRK2 R1441G and VPS35 D620N cells, we performed two additional, independent replicates. The updated compiled data confirm that both variants have elevated levels of LRRK2 on isolated lysosomes at baseline compared to WT cells and show a non-significant trend of a further increase in LRRK2 levels following LLOMe treatment .These data suggest that increased LRRK2 recruitment to lysosomes upon damage may at least, in part, contribute to elevated Rab10 phosphorylation observed in LRRK2 R1441G and VPS35 D620N KI cells with LLOMe treatment. The extent of lysosomal Rab10 phosphorylation is far greater than the extent of increase in LRRK2 levels following LLOMe treatment, suggesting that additional mechanisms beyond increased recruitment of LRRK2 to lysosomes may contribute to this effect on LRRK2 activation as suggested by the reviewer.In addition to updating Figure 4 with this new data, we have also modified the text to remove our conclusion that LLOMe treatment does not further impact LRRK2 levels in PD-linked variant cells and added new text to the results section (as described above) to highlight that additional mechanisms beyond increased LRRK2 recruitment to lysosomes may also contribute to LRRK2 activation following lysosomal damage in these cells.3. Based on data in Figure 4A, it is claimed that lysosome damage causes LRRK2 to accumulate at lysosomes. However, in Figure 4D the same treatment fails to yield a similar increase in LRRK2 levels at lysosomes. These inconsistencies undermine confidence in these results and should be explained.The reviewer astutely points out that Figure 4A shows a significant increase in LRRK2 levels on lysosomes in WT cells following LLOMe treatment, while Figure 4D shows a non-significant trend toward an increase in lysosomal LRRK2 levels under similar conditions. As described above, we think this variability is due to the low levels of LRRK2 detected on lysosomes upon endogenous expression and technical variability in recovery of lysosomes across experiments using the Lyso-IP method.PNAS 2018 PMID: 30209220, Herbst et al EMBO J 2020 PMID: 32643832, and Bonet-Ponce et al Science Adv 2020 PMID: 33177079), and further supported by orthogonal imaging-based studies , increasing confidence that lysosomal damage causes LRRK2 to accumulate at lysosomes.Our observations are consistent with previous work .The reviewer raises a valid concern regarding whether LLOMe treatment might confound our ability to successfully isolate lysosomes following lysosomal membrane permeabilization. As described in more detail in our response to point #4 from Reviewer #1, we consistently observe a depletion in LAMP1 levels on isolated lysosomes following LLOMe treatment while the lysosomal levels of TMEM192-3xHA were minimally impacted. Our hypothesis is that the loss of LAMP1 following lysosomal membrane permeabilization may arise from the redistribution of LAMPs to the cytosol in small vesicles and/or targeted degradation via lysophagy in response to lysosomal damage, as suggested by Lee et al We thank the reviewer for their suggestion to examine the lysosomal localization of TMEM192-3xHA with and without LLOMe treatment to increase confidence in the use of Lyso-IP to isolated damaged lysosomes. We assessed the colocalization of TMEM192-HA and the lysosomal marker LAMP1 treated with vehicle or LLOMe using high-content confocal imaging. We quantified the extent of colocalization between HA-tagged TMEM192 and LAMP1 and confirmed that LLOMe treatment has no impact on the localization of TMEM192 to lysosomes. This new data add confidence in the use of the Lyso-IP method to isolate damaged lysosomes and has been added to Figure 3- figure supplement 1.5. Fig. 3A shows phosphorylated Rab12 at lysosomes but does not show total Rab12 at lysosomes. Showing total Rab12 is required to support claims of regulated recruitment.We agree with the reviewer that it is important to show total Rab12 levels on isolated lysosomes at baseline and following LLOMe treatment to support our claims of regulated recruitment. This data can be found in Figure 3D of our updated manuscript, showing that Rab12 levels are significantly increased on lysosomes following LLOMe treatment and that this signal is lost in RAB12 KO cells to confirm the specificity of our antibody.6. The methods section lacks details for cell line generation and validation. How was Cas9 delivered? How were clones selected? How were mutations in Rab12 assessed?We thank the reviewer for suggesting that we expand on our methods section with f urther details on how our cell lines were generated and validated. Ribonucleoproteins containing the Cas9 protein and synthetic chemically modified sgRNA produced at Synthego were electroporated into the cells using Synthego's optimized protocol. Editing efficiency was assessed upon recovery, 48 hours post electroporation. Specifically, genomic DNA was extracted from a portion of the cells, PCR amplified and sequenced using Sanger sequencing. The resulting chromatograms are processed using Synthego Inference of CRISPR edits software (ice.synthego.com). To isolate monoclonal cell populations, edited cell pools were seeded at 1 cell/well using a single cell printer into 96 or 384 well plates. All wells were imaged every 3 days to ensure expansion from a single-cell clone. Clonal populations were screened and validated using the PCR-Sanger-ICE genotyping strategy to confirm successful gene deletion or editing.We have updated our Materials and Methods section to describe how these cell lines were generated and validated.7. Details are lacking on the source and identity of the TMEM192-3xHA plasmid.We have added additional details on the generation of the TMEM192-3xHA plasmid and the construct itself to the Materials and Methods section.Reviewer #1 (Recommendations for the authors):I was really hoping to move this forward; unfortunately, the response to the reviewer comments is not sufficient as follows.1. The authors were asked to modify the title. As it stands, the title implies that Rab12 is on lysosomes which it is normally not, and that LRRK2 is only active on lysosomes which is also not true. The title is really important and either needs to indicate that Rab12 promotes localization to DAMAGED lysosomes or be otherwise modified.We agree with the reviewer that the title for our manuscript is very important and something we want to make sure best describes the findings from our study. Based on the reviewer’s comment, we had modified our title in our last resubmission to “Rab12 regulates LRRK2 activity by facilitating its localization to lysosomes” to soften our language around the requirement of Rab12 in mediating LRRK2’s localization to the lysosomes. We now appreciate that this did not fully address the reviewer’s concern around the role that lysosomal damage plays in this recruitment. Based on the reviewer’s feedback, we have further modified our title to “Rab12 is a regulator of LRRK2 activity and mediates its localization to damaged lysosomes.” We thank the reviewer for their additional feedback on the title and are happy to further adjust the title as needed.2. LRRK2 still produces pRab10 in Rab12 knockout cells. The authors claim on Page 13 that Rab12 is necessary and sufficient for Rab10 phosphorylation but this is not correct. It is clear that LRRK2 still produces pRab10 in cells lacking Rab12 from blots in 2A and 1D. In Figure 2C, the authors show significant variability in Rab10 levels. This highlights the importance of normalizing phosphoRab10 levels to total Rab10 levels. Please normalize data to total Rab10 and not GAPDH or other protein. This is standard in this field.We appreciate the reviewer’s comments around the requirement of Rab12 for LRRK2-dependent phosphorylation of Rab10, and we have provided new data and further revised the text to address these points. Using our sensitive MSD-based assay to quantify pT73 Rab10 levels under basal conditions, we observed a similar reduction in Rab10 phosphorylation with Rab12 knockdown or deletion as we also observed with LRRK2 knockdown or deletion  which led us to make this comment. As the reviewer noted, we do observe some residual Rab10 phosphorylation upon Rab12 knockdown/deletion when assessed by western blot analysis . This signal is also observed in LRRK2 KO A549 cells, which may be a result of background signal from western blot analysis or may also suggest that a minor proportion of Rab10 phosphorylation may be LRRK2- and Rab12- independent in this cell model as proposed by the reviewer. We appreciate this reviewer’s point and have therefore modified the text to remove any reference to Rab12 being required or sufficient for LRRK2-dependent Rab10 phosphorylation. Specifically, we have changed the subsection heading of the results section on page 13 from “Rab12 is necessary and sufficient for LRRK2-dependent phosphorylation of Rab10" to “Rab12 deletion attenuates LRRK2-dependent phosphorylation of Rab10.”The reviewer also commented on variability in total Rab10 levels across clones examined in Figure 2C and suggested we normalize pRab10 levels to total Rab10 levels. Phospho-Rab10 levels in Figure 2B were measured using a quantitative MSD-based assay and normalized for protein input. Issues with specificity and sensitivity precluded us from using an MSD-based assay against total Rab10, so western blot analysis was used to measure total Rab10 levels. To address the reviewer’s concern, we have now normalized pRab10 levels (quantified using an MSD-based assay) to total Rab10 levels measured using western blot analysis. These new data have been added as a new supplementary figure . These new data confirm our original findings in Figure 2B, strongly suggesting that alterations in Rab10 levels do not explain the significant reduction in pRab10 levels observed in Rab12 KO cells. They are presented on page 13 of the revised manuscript.3. The only data on Rab12 being on lysosomes comes from lyso-IPs. In order to understand the magnitude of the process being studied here the authors need to state the fraction of the total Rab12 and total LRRK2 in the LysoIPs of Fig. 3 and 4. This is essential. For example, it is possible that Rab12 increases LRRK2 on lysosomes from 2% of total to 4% of total. The reader needs this information.We thank the reviewer for these comments and have now added several additional data panels related to them that we feel have strengthened the paper. To address the reviewer’s question regarding the percentage of total LRRK2 and Rab12 present on isolated lysosomes, we quantified LRRK2, Rab12, and HA band intensity in WT cells from both the IP and PNS  samples used for Figure 3D and 4A. We then calculated the total amount of each species present in both sample types by extrapolation based on the amount of sample loaded for western blot analysis and determined the total percentage of each species recovered in the IP. This analysis indicates that ~2% of LRRK2 is on lysosomes at baseline, and LLOMe treatment roughly doubles this to ~4% present on lysosomes. Similarly, ~1% of total Rab12 is present on lysosomes at baseline, and LLOMe treatment increased this to ~1.5% present on lysosomes. This analysis has been added as new data to our updated manuscript in Figure 3- figure supplement 2G and is presented on page 22 of the revised manuscript. Our analysis of total HA recovery in the IP samples confirms that less than 100% of the total HA-labeled lysosomes present in the input were captured in all cases – by experimental design to ensure equal capture across conditions– which ultimately suggests that the calculated total LRRK2 and Rab12 levels present on lysosomes are likely underestimates of the true values.We had previously included imaging data that showed Rab12 localization to lysosomes basally that was significantly increased upon LLOMe treatment . As an orthogonal approach to address the reviewer’s question, we quantified the percentage of LRRK2 and Rab12 on lysosomes at baseline and following LLOMe treatment using imaging-based analysis. Specifically, we overexpressed either fluorescently-tagged LRRK2 or Rab12 and quantified the % of LRRK2 or Rab12 on lysosomes by measuring the amount of LRRK2 or Rab12 signal that colocalized with LAMP1 normalized to the total LRRK2 or Rab12 signal per cell. Our imaging-based analysis suggested that ~5% of LRRK2 localized to lysosomes at baseline and that LLOMe significantly increased this to ~6%, and ~12% of Rab12 was observed at lysosomes basally and ~14% following LLOMe treatment. These data have been added as a new figure panel in Figure 3- figure supplement 2H and is presented on page 22 of the revised manuscript. This analysis required overexpression of both LRRK2 and Rab12 which also has its caveats with respect to recapitulating endogenous localization but nonetheless provides a useful complimentary approach.We employed two distinct methods to estimate the percentage of LRRK2 and Rab12 on lysosomes basally and following LLOMe treatment. Both methods support our conclusions that a portion of total LRRK2 and Rab12 localize to lysosomes and that lysosomal damage further increases their lysosomal localization. The potential caveats associated with both approaches make it difficult in the end for us to be confident about the precise quantification of both proteins on lysosomes. Nevertheless, we do agree with the reviewer that this data addresses an essential question and have therefore decided to include it with the caveat as stated on lines 733-738:“These results also show that a small percentage of Rab12 and LRRK2 are present on lysosomes at baseline and that lysosomal damage leads to a significant increase in the localization of both proteins to the lysosome , but precise quantification of the amount of Rab12 and LRRK2 on lysosomes under these conditions is difficult and warrants further study.”4. The authors were asked to carry out nocodazole experiments to document true lysosome co-localization. 293T cells are poorly adherent and give poor microscopy images with or without LLOME or nocodazole. Why didn't the authors use their A549 cells as in Fig. 3E? Note that a Golgi marker in these cells would have shown complete co-localization with lysosomes in this scenario-this is why nocodazole is so important to separate membrane compartments. The authors need a clear and well controlled experiment showing that the structures they visualize are lysosomes and not Golgi derived vesicles. This circles back to the title of the paper.We thank the reviewer for their suggestion to use nocodazole to confirm the colocalization of LRRK2 and Rab12 and to more clearly understand if LRRK2 and Rab12 are recruited to lysosomes rather than the Golgi upon LLOMe treatment. In line with their suggestions, we have conducted new studies and added new figures to the manuscript that we feel have further strengthened the manuscript.eLife, 2019) and found that reducing the nocodazole treatment to 25 uM for 2 hours reduced toxicity and enabled subsequent analysis. We saw that the Golgi marker GM130 showed fragmented and dispersed punctate structures, confirming the effects of nocodazole treatment in this paradigm.To examine this, we conducted new studies using overexpression of eGFP-tagged LRRK2 and mCherry-tagged Rab12 to follow the localization of each protein at baseline and following lysosomal damage. We had initially tried to use A549 cells, but it turned out to be very challenging to identify a sufficient number of cells expressing both eGFP-LRRK2 and mCherry-Rab12 due to poor transfection efficiency in A549 cells . We therefore used HEK293T cells to address the reviewer’s comment. We further optimized the concentration and duration of nocodazole treatment based on previously-published work  LLOMe treatment significantly increased the colocalization of Rab12 with the lysosomal marker LAMP1 but not with the Golgi marker GM130 (as quantified by Pearson’s correlation coefficient). This co-localization was retained upon nocodazole treatment . These data support our hypothesis that lysosomal damage increases the localization of Rab12 to lysosomes and not Golgi-derived vesicles.2) LLOMe treatment also significantly increased the colocalization of LRRK2 with LAMP1 but not with the Golgi marker GM130 (quantified by Pearson’s correlation coefficient). As was observed with Rab12, this co-localization was retained upon nocodazole treatment . These data add confidence to our conclusions that lysosomal damage increases the localization of LRRK2 to lysosomes.3) To add confidence that Rab12 and LRRK2 colocalize following LLOMe treatment, we quantified their colocalization using Pearson’s correlation coefficient. We observed colocalization between Rab12 and LRRK2 following lysosomal damage and saw that this co-localization was preserved upon nocodazole treatment (new figure 3F). These data further support our conclusion that Rab12 and LRRK2 colocalize with one another following LLOMe treatment.Overall, these data add confidence that a portion of LRRK2 and Rab12 localize to lysosomes a baseline, LLOMe treatment induces increased localization of both LRRK2 and Rab12 to lysosomes but not to the Golgi, and LRRK2 and Rab12 colocalize following lysosomal damage. We thank the reviewer for their suggestion that has further strengthened our conclusions.Reviewer #2 (Recommendations for the authors):The authors have responded to all of the concerns raised. It is nice to see that LRRK2 is not required for the localization of rab12 to the lysosome. What is very clear is that LLOMe recruits rab12 to the lysosome, induces phosphorylation of rab10 and rab12 is required for phosphorylation of rab10. However, a discrepancy remains between the recruitment of LRRK2 to the lysosome by LLOMe, which is not as apparent for wt LRRK2 in Fig. 4D as in 4A, and the phosphorylation of rab10 induced by LLOMe . So it seems likely, as the authors have now included in the results, that mechanisms other than lysosomal recruitment of LRRK2 contribute to the phosphorylation of rab10. Importantly, they have also shown that LRRK2 is responsible for the induction by LLOMe , so I think this is fine, but they should probably include this important consideration in the discussion as well as the results.We are pleased that our previous submission addressed all of the concerns raised by this reviewer and thank them for their suggestions that have substantially improved our manuscript. We agree that additional mechanisms beyond lysosomal recruitment of LRRK2 may contribute to the phosphorylation of Rab10. In addition to the text that was added in the results section around this in our last submission, we have updated the discussion to also include this important consideration as suggested by the reviewer. Specifically, we have added the following text to the Discussion (lines 908-915):“We cannot rule out that additional mechanisms beyond increased proximity between LRRK2 and its Rab substrates may contribute to Rab10 phosphorylation following lysosomal damage, as the magnitude of Rab10 phosphorylation induced by LLOMe treatment was greater than the extent of LRRK2 recruitment to damaged lysosomes. Additional studies are warranted to determine how lysosomal membrane rupture triggers Rab12 recruitment, to identify other regulatory processes that may contribute to Rab phosphorylation upon lysosomal damage, and to better define how broadly such mechanisms are employed to drive LRRK2 activation in PD.”Reviewer #3 (Recommendations for the authors):The revised manuscript provides strong evidence that Rab12 can regulate the ability of LRRK2 to phosphorylate Rab10 and that this occurs at least in part by regulating the abundance of LRRK2 at lysosomes. Although key questions about the mechanism remain unanswered, these are still important findings. New data was added to the manuscript that helped to address concerns that were raised during the previous review. Below I have provided some suggestions for clarification to the text that should be addressable without the need for new experiments. However, I also remain concerned about some important results and their interpretation (points 1-).1. There is a discrepancy between the strong decrease in LAMP1 levels in the lysosome IP samples in Figure 3A and that is not seen in the LAMP1 immunofluorescence signal in 3C. This should be acknowledged and/or explained in the text.The reviewer correctly notes that we did not observe a significant decrease in LAMP1 signal assessed via imaging following LLOMe treatment, in contrast to the decrease observed in isolated lysosomes. LLOMe treatment leads to permeabilization of the lysosomal membrane, and these data may suggest that we are immunopurifying fragments of lysosomal membrane from which LAMP1 has been dissociated or perhaps degraded. We observed strong recruitment of Gal3 to isolated lysosomes following lysosomal damage, supporting some membrane integrity and glycocalyx remain after LLOMe treatment. Our imaging-based analysis did not show obvious differences in LAMP1 signal with LLOMe treatment, likely because our fixation and imaging conditions would capture lysosomal membrane fragments and dissociated LAMP1.To acknowledge this point, we have added the following text to the results section (lines 287-295):“While LLOMe treatment reduced the levels of lysosomal-associated membrane protein 1 (LAMP1) in isolated lysosomes, the levels and localization of TMEM192-3xHA, the lysosomal membrane protein used to isolate lysosomes, were not significantly impacted by LLOMe treatment . We did not observe a loss of LAMP1 signal by immunofluorescence analysis, suggesting that LAMP1 may dissociate or be degraded from ruptured lysosomal membranes upon immunopurification. These data suggest that while LLOMe treatment results in lysosomal membrane damage, sufficient lysosomal integrity remains to enable purification of this subcellular compartment using TMEM192.”2. Lines 272-275 imply that previous studies (references 32 and 33) also saw reductions in LAMP1 levels on purified lysosomes following LLoMe treatment. However, I could not find evidence of this in the papers that were cited.We appreciate that the inclusion of references 32 and 33 in our previous draft following our comment around potential explanations for the reduction in LAMP1 levels observed on isolated damaged lysosomes was not clearly elaborated. References 32 and 33 had been previously included to support our hypothesis that LAMP1 levels may be reduced due to enhanced lysophagy or redistribution to small vesicles upon damage. These papers showed that LLOMe treatment led to the redistribution of LAMP2 to the cytoplasm or increased the turnover of LAMP1, respectively. However, based on the great point raised by this reviewer that LAMP1 signal loss was not observed in our imaging-based studies, our speculation is that we are immunopurifying lysosomal membrane fragments from which LAMP1 has dissociated or been degraded. We have revised the text as described above in Point 1 to better reflect our thinking on this and removed reference to these papers entirely in the updated manuscrpt.3. There is still a discrepancy between Figure 4A-C where LLoMe activates LRRK2 (as measured by phosphor-Rab10) even in the context of VPS35-D620N and LRRK2-R1441G and Figure 4D where LLoMe does not change LRRK2 lysosome abundance in the same cells. The authors touched on this in the response to reviewers but the answer was not definitive. The description and interpretation of these results in the manuscript text is confusing as it is unclear to what extent LRRK2 kinase activity is regulated in response to these stimuli versus LRRK2 localization and proximity to substrates. The data suggests that there might be independent changes to LRRK2 localization and activity. It is not necessary for the authors to solve these puzzles but they should be more clearly presented.We sincerely appreciate the reviewer’s feedback that the description and interpretation of our results around the magnitude of increase in Rab10 phosphorylation being greater than the increase in LRRK2 localization to lysosomes observed following LLOMe treatment should be edited to more clearly represent our ideas on the balance between activity versus localization. To further clarify our results and interpretation of our data, we have modified our results section to the following (lines 849-857):“The levels of LRRK2 on lysosomes were significantly increased in untreated LRRK2 R1441G KI cells and VPS35 D620N KI cells, suggesting that enhanced localization of LRRK2 to lysosomes and proximity to its Rab substrates may contribute to the elevated Rab10 phosphorylation observed on lysosomes at baseline in these cells . Rab10 phosphorylation was increased on lysosomes in response to LLOMe treatment while the levels of LRRK2 were not significantly impacted on lysosomes isolated from LRRK2 R1441G and VPS35 D620N KI cells, suggesting additional mechanisms beyond LRRK2 localization may also contribute to LRRK2 activation in response to lysosomal damage in these cells.”Further, we have added the following text to the Discussion (lines 908-915):“We cannot rule out that additional mechanisms beyond increased proximity between LRRK2 and its Rab substrates may contribute to Rab10 phosphorylation following lysosomal damage, as the magnitude of Rab10 phosphorylation induced by LLOMe treatment was greater than the extent of LRRK2 recruitment to damaged lysosomes. Additional studies are warranted to determine how lysosomal membrane rupture triggers Rab12 recruitment, to identify other regulatory processes that may contribute to Rab phosphorylation upon lysosomal damage, and to better define how broadly such mechanisms are employed to drive LRRK2 activation in PD.”4. Line 168: The statement that Rab12 is necessary and sufficient is too strong. Some commentary is required to at minimum acknowledge that this study only examined A549 cells and that the extent that the findings are generalizable to other cell types remains unknown.Based on the reviewer’s suggestions, we have removed our statement regarding Rab12 being necessary and sufficient. Specifically, we have revised the text in the results section (line 165 on page 10) from “Rab12 is necessary and sufficient for LRRK2-dependent phosphorylation of Rab10” to “Rab12 deletion attenuates LRRK2-dependent phosphorylation of Rab10.” Further, we have added the following text to the Discussion (lines 915-918 on page 31):“As our work focused on the role of Rab12 in A549 cells, it will also be important to understand whether Rab12 similarly regulates LRRK2 activation and localization to damaged lysosomes in other cell types.”5. Figure 3A and D and Figure 4A-D should also show the total cell lysates rather than just the IP results.We thank the reviewer for their suggestion to include the results from total cell lysates in addition to our IP results. We have included side by side blots showing the western blot data from the immunoprecipitated samples as well as the PNS . These have been added to Figure 3- figure supplement 1B and F and Figure 4-figure supplement 1A-D.6. Figure 4E and Line 505 over-reach in stating that Rab12 mediates LRRK2 recruitment to lysosomes. Proving this would require evidence that a direct interaction between LRRK2 and Rab12 is necessary for LRRK2 recruitment and activation.We appreciate the reviewer’s point regarding softening our language around Rab12 mediating LRRK2 recruitment to lysosomes without direct evidence that an interaction between these proteins is required for LRRK2 recruitment to damaged lysosomes. We have revised Figure 4E from “Rab12 mediates LRRK2 recruitment and Rab phosphorylation on lysosomes” to “Rab12 regulates LRRK2 recruitment and Rab phosphorylation on lysosomes.” Further, we have revised the sentence in the discussion to remove “mediates” and instead use the word “regulates”:“Our findings provide key insight into the mechanism by which LRRK2 activity is increased in response to lysosomal damage by demonstrating that Rab12 regulates LRRK2 localization to ruptured lysosomes.”


Legumes manage both symbiotic (indirect) and non-symbiotic (direct) nitrogen acquisition pathways. Understanding and optimising the direct pathway for nitrate uptake will support greater legume growth and seed yields.2-fixation pathway involving soil-borne rhizobia bacteria, the acquisition of nitrate and ammonia from the soil can also be an important secondary nitrogen source to meet plant N demand. The balance in N delivery between symbiotic N (indirect) and inorganic N uptake (direct) remains less clear over the growing cycle and with the type of legume under cultivation. In fertile, pH balanced agricultural soils, NO3− is often the predominant form of reduced N available to crop plants and will be a major contributor to whole plant N supply if provided at sufficient levels. The transport processes for NO3− uptake into legume root cells and its transport between root and shoot tissues involves both high and low-affinity transport systems called HATS and LATS, respectively. These proteins are regulated by external NO3− availability and by the N status of the cell. Other proteins also play a role in NO3− transport, including the voltage dependent chloride/nitrate channel family (CLC) and the S-type anion channels of the SLAC/SLAH family. CLC’s are linked to NO3− transport across the tonoplast of vacuoles and the SLAC/SLAH’s with NO3− efflux across the plasma membrane and out of the cell. An important step in managing the N requirements of a plant are the mechanisms involved in root N uptake and the subsequent cellular distribution within the plant. In this review, we will present the current knowledge of these proteins and what is understood on how they function in key model legumes . The review will examine their regulation and role in N signalling, discuss how post-translational modification affects NO3− transport in roots and aerial tissues and its translocation to vegetative tissues and storage/remobilization in reproductive tissues. Lastly, we will present how NO3−influences the autoregulation of nodulation and nitrogen fixation and its role in mitigating salt and other abiotic stresses.Legumes have multiple pathways to acquire reduced nitrogen to grow and set seed. Apart from the symbiotic N The majority of HATS genes and encoded proteins are activated when soil NO3− concentrations are low    to be utilised. The transport process is mostly an energy dependent (active) process  has been linked to a NO3− efflux activity in response to ammonium (NH4+) toxicities and the subsequent acidification of the apoplast, while the NO3−/peptide transporter NAXT allows for passive NO3− efflux across the PM in response to increased acidities around the roots  where homeostatic balance in N supply (direct supply) is offset against the N-dependent regulatory controls of the biological N2-fixing or acquisition symbiosis.The diversity of physiological responses to NON depleted soils via symbiosis with compatible soil rhizobia. The symbiosis results in the development of root nodules that house N2-fixing bacteroids within a cellular environment conducive to the fixation of atmospheric N2 to NH3  to support their N needs independent of the existing symbiosis. Furthermore, high levels of NO3− and or ammonium (NH4+) in the soil actively inhibits symbiotic N2-fixation fixation (SNF), which could be due to the activity of NO3− and NH4+ (AMT) transporters in legume roots and nodules . This dichotomy in N acquisition strategy provides flexibility to legumes to secure N to meet plant demand and to adjust to fluctuations in the availability of N in the soil. However, this also creates difficulties in managing the symbiotic partnership, which is negatively impacted by the concentration of reduced N in the soil  inhibition. There were differences in inorganic N uptake among the different legume species in field experiments, which was positively correlated to rapid lateral root expansion and soil colonisation  and their involvement in NO3− transport in plants and will unravel those activities previously characterised in the model legumes, Medicago truncatula (Medicago), Lotus japonicus (Lotus) and Glycine max (soybean). The review will then explore signalling activities of these transporters for root development and nodulation, their post-translational regulation and role of these transporters in legume nodules and their influence on N2-fixation, and nodule N homeostasis. Tissue NO3− transport, storage and redistribution will be investigated and their role in the mitigation of different abiotic stresses. We hope this review will highlight where the research gaps exist in this field and where future research is required to better understand this alternative N uptake pathway operating in all legumes.The rate of NO3− transporters are divided into four general classifications. The first is the large Nitrate Transporter 1/Peptide Transporter (NRT1/PTR/NPF) family , maize  and tomato (LeNrtl-l and LeNrtl-2)  NO3− transporter genes  was expressed during seed development. In Glycine soja, expression of GsNRT1.57, GsNRT1.96 (NPF 7), GsNRT1.84 (NPF1) are induced by N supply  enhances NO3− flux into Xenopus laevis oocytes. In plants, MtNPF6.8 expression in roots is enhanced when NO3− is absent in the growth media and repressed when present. MtNPF6.8 is also considered a transceptor by its ability to also mediate ABA transport and regulate primary root growth , which shows higher nodulation sensitivity to NO3− than WT plants. Mtnip-1 contains a lesion in the gene MtNIP/LATD (MtNPF1.7), which is an NPF ortholog  show defects in nodule vasculature development and a reduction in bacteroid nitrogenase activity. MtNPF7.6 is suggested to mediate NO3− uptake from the soil solution to deliver low concentrations to developing nodules through xylem to phloem transfer in vascular tissues. The rate and quantity of NO3− transport into nodules influences nodule development, possibly through mechanisms involving NO influence of LB activity and oxygen delivery to the bacteroids  showed defective nodule development  show a reduction in N2-fixation but no change to nodulation or nodule number. LjNPF3.1 is expressed in roots and in the nodule outer cortex . Shoot growth in the mutant was compromised but could be recovered at elevated NO3− supply (5 mM).The low-affinity NO3− transport (HATS) in plants. NRT2 transporters share structure similarity with NPFs having 12 transmembrane regions with a large hydrophilic loop between TM 6 and TM 7, although both families do not share sequence homology  was higher in plants grown with NO3− compared to N-deprived conditions. The expression of GmNRT2.1 and GmNRT2.2 has since been shown to be in the exodermis and epidermis of soybean roots, a similar expression pattern to AtNRT2.1 and AtNRT2.4 . Two NRT2 genes (LjNRT2.1 and CM0161.180 (LjNRT2.7)) showed strong induction while the other two genes (LjNRT2.2 and CM0001.20) showed no response to NO3−  under both symbiotic and non-symbiotic conditions, Ljnrt2.4 mutants displayed significant reduction in shoot biomass, NO3− content and nitrogenase activity in nodules compared to the inoculated wild type. In Medicago, three NRT2 genes  have been identified with varied expression in roots, shoots and nodules  under minus N conditions or when supplied NO3−   except for AtNRT2.7  . CLC genes have been found in various plants with seven CLC homologues identified in Arabidopsis  has shown similar affinities for NO3− and Cl− anions. GcCLC-c2’s affinity for the Cl− anion is pH independent as compared to GmCLC1, where Cl− affinity was pH dependent  was downregulated alongside other NO3− transporter related genes (LjNRT2.1 and LjNRT2.1) in nodulated roots compared to roots without rhizobia inoculation  or suggests a nodulation enhanced repression of gene activity. It will be important to further explore this family to examine their potential involvement in salt tolerance and/or drought and their relationship to root symbiotic N status and whether resupply of NO3− to nodulated roots would activate their expression and activity.In legumes, GmCLC1 is located on the tonoplast and is induced by water and NaCl stress in soybean leaves and roots  have been shown to be nitrate transporters and located on the symbiosome membrane at late stage of nodule development. Both proteins are not related to NPF or NRT2 and are able to transport both NO3− and Cl−1 . NO3− sN concentrations. NO3− can also deregulate the rhizobia symbiosis and impact both root and nodule development in positive (low concentrations) or negative (high concentrations). The Medicago AtNPF6.3 ortholog, MtNPF6.8 has been reported as master nitrate signal sensor in primary root tips  of primary root growth in a Mtnpf6.8 mutant  has also been reported to have a role in ROS homeostasis  might be involved in ABA signalling. In addition to slow root growth and development, the latd mutant also showed abnormal nodule development, with infection thread arrest in root hairs with a rhizobial deprived primordium. ABA application fails to rescue the latd phenotype, suggesting that the nitrate induced nodule regulation by MtNFP1.7 is ABA independent  transcription factors are involved in regulating gene expression of NO3− transporters and assimilation genes  belonging to RWP-RK family of plant transcritiopn factors have emergered as major players of NO3− signalling pathways by activating the expression of root derived CLE peptides. Nod factor induces NIN expression which transcriptionally regulates up to three CLE genes, CLE-RS1/2/3 in roots. The encoded peptides are mobile signals that interact with respective receptors in shoots of different legume species including, the Lotus HYPERNODULATION ABERRANT ROOT1 (HAR1)  and Medicago   called NO3− responsive elements (NREs) located in the promoters of genes, including NRT2.1 and NIR1 , accumulated NO3- promotes the binding of NLP1 to the promoter of NRT2.1, which further increases NRT2.1 expression and consequently NO3- influx into the cell. The increased NO3− concentration in the cytosol triggers a nuclear enrichment of NLP4 which can then activate the expression of NRE regulated CLE-RS2 genes and the onset of the AON regulatory pathway   receptor. In Medicago, the high-affinity nitrate transporter, MtNRT2.1 has been reported recently in regulation of nodulation  and yield related traits  thus negatively regulating the primary NO3− response under low NO3− concentrations, while in contrast CIPK8 kinase acts a positive regulator for the low-affinity NO3− response  have conserved Ser residues at or near the same position in all NRT members  and ureides exported from determinate nodules .In most legumes, N to different sections of the nodule is important for maintaining N homeostasis and for the delivery of N out of the nodule to support growth for the rest of the plant  and nodule development. One mechanism was proposed where NO3− derived NO competitively influences the ability of leghemoglobin to bind oxygen and disrupt the delivery of oxygen to actively respiring bacteroids  is also expressed in vascular  cells  show defects in nodule vasculature development and a reduction in bacteroid nitrogenase activity, possibly through an accumulation of NO and a reduction in leghemoglobin expression. MtNPF7.6 is suggested to mediate NO3− uptake from the soil to deliver low concentrations into developing nodules through a xylem to phloem vascular transfer. A similar mechanism was reported in rice which showed OsNPF2.2’s role in vasculature development of different organs in rice , has a role in nodulation where mutant plants (Mtlatd) produce defective nodules , shoot NO3− content increased as did leaf area and shoot growth. The disruption of basipetal NO3− transport did not impact the negative influence of NO3− on legume nodulation but also didn’t disrupt N2-fixation capacities of the nodules  is expressed in the leaf petiole. AtNRT1.4 is a low-affinity NO3− transporter which when inactivated reduces the NO3− content in the petiole while increasing in the leaf lamina. Petiole NO3− content has been used to monitor N fertiliser demand in some plants  in Glycine soja leaves raises the possiblity that these transporters are involved in leaf NO3− homeostatis. Further investigations are required to explore their role in the recycling of N metabolites in plants.AtNPF4.6 (AtNRT1.2) transports both NON supply also motivates plants to transfer nutrients from older to younger leaves to support their growth. Both low and high-affinity transporters are involved in this remobilization. In this context, AtNPF2.13 (NRT1.7) mobilises nitrate from old to young leaves via phloem loading  also appear to influence the transfer of NO3− from old to young leaves (Hsu and Tsay AtNRT2.4 and AtNRT2.5) may also participate in the phloem loading and translocation of NO3− to aerial tissues. The N inducible, AtNRT2.4 is expressed in the phloem of leaves and its loss of activity reduces leaf NO3− content  is expressed in vascular tissues of siliques and in the funiculus suggesting a role in delivering NO3− to developing seeds. Early NO3− delivery was found to be important at the one to four cell stage of early embryogenesis where loss of supply resulted in abnormal embryo development  has also reported to be involved in NO3– transport into the embryo at the bent cotyledon stage of developing seeds . The transgenics grew better with significantly higher relative leaf water content and less relative electrolyte leakage than observed in WT plants. Moreover, the concentration of Cl− ions in the roots of transgenic plants was lower than the controls , a process which can influence external pH  to make it NO3− selective  was less prominent. This indicated Cl− uptake activity for both MtNPF’s, with MtNPF6.5 like ZmNPF6.4 as could behave as a Cl− selective transporter. Under salt stress, mtnpf6.5–3 mutants showed reduced Cl− contents in roots and shoots (48–55% and 22–26%) than WT while mtnpf6.7 mutants showed Cl− levels to the WT. Longer primary roots with more lateral roots were developed in the Mtnpf5.6 and Mtnpf6.7 mutants under salt stress. However, in the presence of NO3− these phenotypic changes were abolished. Recently, the expression of GsNRT2.3, GsNRT2.4, GsNRT1.12, GsNRT1.43, GsNRT1.62 and GsNRT1.57 were found to be upregulated when Glycine soja plants were treated with alkaline salts (NaHCO3)  have been observed in wheat roots (TaNRT2.1)  and MtNRT3.1L2 (MTR_4g104730) help decrease arsenic (As) contamination in plants  declined when the expression of MtNRT3.1 was downregulated. Furthermore, complementation of MtNRT3.1L1 in nrt3.1 mutants showed that NRT3.1 alone or via NRT2.1/NRT3.1 confers As (V) tolerance.Heavy metal contamination poses a great threat to human health due to the potential absorption and incorporation into the food chain  have greatly expanded our knowledge of the processes managing NO3− transport to support both growth and seed development. The relationship between alternative N acquisition systems (N2-fixation and direct root uptake) are slowly starting to take shape identifying shared signalling pathways managing root and nodule development and the interdependency on alternative reduced N reserves to support early development of legumes subject to a rhizobial inoculation. The important next steps will be to define the regulatory controls limiting both nodulation and soil N to allow these systems to be used effectively together without penalties linked to carbon availabilities. Further work is required to understand the different layers of feedback mechanisms controlling N assimilation and the spatial (cell types) and temporal time periods they operate under. Genetic resources are improving in legumes, which will result in a rapid expansion of knowledge in this space to support the further growth and utilisation of legumes for sustainable protein production and the indirect benefits, through N deposition in the soil, weed and disease management afforded to other crops grown in rotation with legumes.Studies across several model plant systems (ZR. and BNK conceptualised the idea, ZR, MNS and BNK performed the literature survey and wrote the draft manuscript, FP-W provided necessary suggestions, ZR, FP-W and BNK edited and critically evaluated the draft manuscript prior to submission.


Trem2 expression levels, there is more data available on how the human Trem2 gene functions in the pathophysiology of AD.Recent genetic studies have highlighted the central role of the innate immune system in Alzheimer’s disease (AD) by identifying several risk variants in genes that are exclusively expressed within microglia in the brain. Most notably, rare variants, such as the R47H, R62H and H157Y mutations, in the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) gene increase risk for late-onset AD [Molecular Neurodegeneration, Tran et al. and Qiao et al. push the field of TREM2 biology forward through two uniquely and newly generated mouse models. Tran et al. explore the response of a new Trem2 R47H knock-in mouse model without cryptic splicing to acute demyelination following administration of cuprizone and within the context of early and late amyloidosis [Trem2 H157Y knock-in mouse model to study the role of the H157Y variant within the context of amyloidosis [In two research articles recently published in loidosis . Qiao etTrem2R47H mouse models, including the use of CRISPR/Cas9 and through bacterial artificial chromosome (BAC) technology. It has been reported that Trem2R47H knock-in models created through CRISPR exhibit aberrant splicing and reduced expression levels of Trem2 mRNA [Trem2 mRNA expression, but similar levels of human TREM2 protein in myeloid cells, compared to CV mice [Trem2R47H NSS  model compared to the Trem2R47H CSS (Cryptic Splice Site) model display expression of Trem2 at similar levels to wild type (WT) mice without evidence of cryptic splicing products  plaques in 5XFAD mice and showed that Aβ levels and compaction are brain region-, age- and sex- specific in Trem2R47H NSS mice. Largely, they showed that the Trem2R47H NSS mice exhibited suppressed microglial activation early in disease that conversely shifted to a unique interferon signature with age. The earlier stage also showed impaired microglia-plaque interactions and less compacted plaques with increased dystrophic neurites. This impairment is lost at 12 months of age  Trem2H157Y/H157Y mice exhibit increased shedding of TREM2 as they displayed higher levels of soluble TREM2 (sTREM2) in serum and brain compared to the heterozygous (het) and WT groups. The hom Trem2H157Y/H157Y mice displayed enhanced Aβ clearance and reduced total Aβ burden at the late stage of Aβ development, but minimal changes in the early stage of Aβ deposition. Interestingly, there were no changes in synaptic markers like synaptophysin but an enhancement in paired-pulse facilitation and strengthened long-term potentiation recordings in the hom Trem2H157Y/H157Y mice compared to WT mice. This data supports a beneficial effect of the H157Y mutation on synaptic plasticity, presynaptic function, and on amyloid burden during the later stage of amyloidosis (Fig. Trem2H157Y/H157Y mice have reduced gliosis and decreased Trem2 signaling compared to WT mice.In vivo understanding of a more rare Trem2 mutation the patient carries as not all have the identical effects on AD pathology. As Qiao et al. demonstrate, the Trem2 H157Y mutation did not affect Aβ pathology at the early stage of Aβ development but reduced Aβ burden and increased Aβ clearance at the later stage of amyloidosis. The in vivo effects of the H157Y mutation on Aβ burden differ from what has been observed in vivo utilizing amyloidosis models with the R47H mutation [Trem2R47H NSS mice exhibit suppressed microglial reactivity early in amyloidosis that conversely shifts to a unique interferon signature with age and amyloid accumulation. These effects at the early and later times assessed by Tran et al. also differed based on brain region with noticeable sex differences.Both studies indicate that identifying the optimal time window to intervene in a disease like AD will be key to reach treatment effectiveness when targeting TREM2 function. Additionally, moving forward we may need to consider the specific type of mutation , 9. FurtThese two studies and others also highlight the importance of understanding the type of pathology and pathology severity in the brains of patients with AD when therapeutically targeting TREM2 function. Neuroanatomically, fibrillar Aβ deposition occurs first and most severely in regions such as the precuneus and frontal lobes  during tTrem2H157Y/H157Y mice displayed increased production of sTREM2. It is certainly possible that the increase in sTREM2 is leading to some of these effects. What are the roles of TREM2 and sTREM2 on neuronal activity and function. How will the H157Y mutation and Trem2R47H NSS model affect tau pathology and neurodegeneration? While these and other critical questions remain, the findings from these two exciting papers solidify the growing idea that the effects TREM2 and microglia are context dependent and differ based on disease severity, brain region, age, sex, mutation type and the types of pathologies present. All these factors need to be better studied and considered when designing TREM2 targeting therapies with the goal of mitigating AD and other CNS disease associated pathologies.Overall, the authors’ discoveries provide important new considerations to our approaches for therapeutically targeting TREM2, open new avenues to consider in advancing AD therapeutics and raise several interesting questions for further study. What is it about the H157Y mutation and relationship between microglia, TREM2, sTREM2 and Aβ that results in decreased Aβ when assessed at a late stage of disease? Qiao et al. show that the hom


Poststreptococcal glomerulonephritis (PSGN) is caused by prior infection with specific strains of group A streptococcus (GAS). PSGN is commonly characterised by significant complement C3 depression and maintained C4 levels, especially in children. The 2022-23 GAS outbreak has been associated with childhood morbidity and mortality in the UK due to invasive GAS infections but no PSGN outbreak has been described.st, 2019 and March 31st, 2023 and for patients under the age of 18 at the time of the request. We collected demographic information as well as concurrent anti antistreptolysin and anti-DNAase B antibody levels. We excluded 4 individuals who had only marginally decreased C3 levels .We reviewed the laboratory requests for complement C3 and C4 measurements received by the main East of England immunology laboratory for the 48 months between April 1We assigned cases as possible PSGN if C3 was low, C4 was normal and antistreptolysin serologies were not obtained. We assigned cases as probable PSGN if C3 was low, C4 was normal and antistreptolysin and anti-DNAase B antibody levels were elevated.24 unique patients aged 0-18 were identified (18 boys and 6 girls), 8 possible and 16 probable PSGN cases. All but two of the probable cases were boys. There were no cases of possible or probable PSGN between 9.3.20 and 20.3.22. Half of the observed cases have been observed within the last 12 months, mirroring the reported invasive GAS incidence in England.Table of possible and probable Post Streptococcal Glomerulonephritis cases, East of England Immunology LaboratoryPSGN is unusual in industrialized countries. The monitoring of C3 and C4 complement level requests suggests a possible undiagnosed concurrent to the GAS PSGN outbreak in the UK and the circulation of possible nephritogenic strains.All Authors: No reported disclosures


Figure 4A since both figures were derived from the same experiment. The original There was an error in Results section, subsection PPI inhibits the growth of gastric cancer cells in vitro and induces their ferroptosis: “In addition, using a specific fluorescent probe, FerroOrange, we revealed that PPI increased the levels of ferrous ions in both AGS and MKN-45 cells (A correction has been made to the 45 cells ” is corr45 cells .”Results section, subsection PPI downregulates NRF2 and FTH1 in the gastric cancer cells: “Using Western blot assays to detect ferroptosis-related proteins in the cancer cells after PPI treatment in vitro, we found that PPI downregulated the expression of both NRF2 (in vitro, we found that PPI downregulated the expression of both NRF2 and FTH1 (A correction has been made to the oth NRF2  and FTH1oth NRF2  in both and FTH1  in both and FTH1 , indicatThe corrected