All experimental protocols were approved by the Western University Animal Care Committee (AUP #2017-135) in accordance with the Canadian Council on Animal Care. The tissues used in these experiments were generated and collected during previous work and can be found detailed in Hintermayer et al. (2019). In short, three-month-old Sprague Dawley (Charles River) rats were subject to a single controlled cortical impact injury (CCI) (3.5 m/s, 2.0 mm depth, 500 ms dwell time). Brain lysates and fixed brain tissue were generated during this previous study.

Three animals from each time point were used for immunohistochemical analysis. Animals were perfused with heparin-saline (0.9% NaCl), fixed with 4% paraformaldehyde and 6 μm sections mounted on glass microscope slides.

For routine immunohistochemistry, antigen retrieval was performed in 10 mM sodium citrate (0.05% Tween 20, pH 6.0) using a pressure cooker. Slides were cooled, washed in PBS, and endogenous peroxidase quenched with 3% hydrogen peroxidase in PBS for 5 min. Slides were then washed in PBS and blocked in 5% bovine serum albumin (BSA) for 1h at RT in 0.3% Triton-X in PBS. Primary antibody incubation against LRRK2 (1:1000, Abcam #133474) was performed at 4°C overnight in blocking solution. Slides were then washed in PBS and incubated with biotin-conjugated Rabbit IgG secondary antibody (1:200, Vectastain Elite ABC Kit Peroxidase #PK-6101) diluted in blocking solution for 1h at RT. As per the manufacturer’s protocol, slides were incubated with the Vectastain ABC reagent for 30 min. The antigen target was visualized with 3,3′-diaminobenzidine (DAB). Counterstaining was performed with Harris’ hematoxylin. Individual images were captured on an Olympus BX45 light microscope (Olympus Life Sciences), and a Leica Aperio AT2 microscope slide scanner (Leica Biosystems) captured tile scan images.

For immunofluorescence staining, antigen retrieval was conducted with Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, 0.05% Tween 20, pH 9.0) in a pressure cooker. Slides were cooled and washed in PBS, then blocked (blocking solution: 3% BSA, 0.1 M glycine, 0.25% Triton X-100 in PBS) for 1 h at RT. Primary antibodies against LRRK2 (1:500, Abcam #133474), NeuN (1:500, Sigma-Aldrich #MAB377), GFAP (1:200, BD Biosciences #556330) or Iba1 (1:500, Sigma-Aldrich #MABN92) were diluted in blocking solution and incubated overnight at 4°C. Slides were then washed in PBS and incubated with Alexa Fluor-conjugated secondary antibodies: Donkey anti-Rabbit Alexa488 (1:1000, Thermo Fisher Scientific #A21206) or Goat anti-Mouse Alexa633 (1:200, Thermo Fisher Scientific #A21050) in blocking solution for 1 h at RT, washed in PBS, counterstained with Hoechst 33258 (1:1000, Biotium #40045), washed in PBS and coverslipped with Immuno-mount (Fisher Scientific #9990402). Images were captured on a Leica SP8 confocal microscope (Leica Microsystems), and deconvolution post-processing was completed using Fiji (Schindelin et al., 2012).

We quantified the cell-specific (neuronal, microglia, astrocytic) expression of LRRK2 near the site of injury by counting the number of NeuN⁺, Iba1⁺ or GFAP⁺ LRRK2 immunoreactive cells per 180 μm². Only cells with a defined nucleus were included for the quantification. Three representative images for each animal were taken at 63× and averaged. A total of three animals were analyzed per timepoint and condition. For neurons, in addition to the percentage of neurons expressing LRRK2, we described the pattern of cellular somatotopic immunoreactivity.

eGFP-WT 2N4R human tau has been previously described (Gohar et al., 2009). 2XMyc-LRRK2-WT (Addgene plasmid # 25361; http://n2t.net/addgene:25361; RRID:Addgene_25361; WT LRRK2), 2XMyc-LRRK2-G2019S (Addgene plasmid # 25362; http://n2t.net/addgene:25362; RRID:Addgene_25362; G2019S LRRK2), 2XMyc-LRRK2-3XKD (Addgene plasmid # 25366; http://n2t.net/addgene:25366; RRID:Addgene_25366; 3XKD LRRK2) and 2XMyc-LRRK2-K1906M were gifts from Mark Cookson (Greggio et al., 2008; Chia et al., 2014). eGFP-Rab10 was a gift from Marci Scidmore (Addgene plasmid # 49472; http://n2t.net/addgene:49472; RRID:Addgene_49472) (Rzomp et al., 2003).

HEK293T (ATCC #CRL3216) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 0.5% penicillin-streptomycin (Invitrogen) at 37°C and 5% CO₂. Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were transiently co-transfected with eGFP-WT 2N4R tau, and 1, 2 or 4 μg of LRRK2-WT, LRRK2-G2019S, LRRK2-3XKD or LRRK2-K1609M to examine LRRK2’s effect on tau phosphorylation. HEK293T cells were also transfected with LRRK2-WT, LRRK2-G2019S, LRRK2-3XKD or LRRK2-K1609M and eGFP-Rab10 to confirm LRRK2 activity. All transient transfections were allowed to express for 48 h.

At 48 h post-transfection, cells were washed briefly on ice with ice-cold PBS and then lysed using NP40 lysis buffer (50 mM Tris pH8.0, 100 mM sodium chloride, 1 mM EDTA pH 8.0, 1% NP40, 10% glycerol) with Halt^™ Protease Inhibitor Cocktail (Thermo Fisher Scientific #78438), sonicated and then pre-cleared (10,000 g, 4°C, 20 min). Protein concentration was determined using DC Protein Assay (BioRad #5000112) as per the manufacturer’s protocol.

For Western blots, equal amounts of total protein were mixed with 5× Laemmli buffer and denatured at 95°C for 10 min. Protein separation was performed using 6%–10% SDS-PAGE with subsequent transfer to 0.2 μM nitrocellulose membrane (Bio-Rad #1620112). Membranes were briefly washed with Tris-buffered saline with 0.1% Tween 20 (TBST) and then blocked with 5% BSA in TBST for 1 h. Primary antibodies against LRRK2 (1:1000; Abcam #ab133474), pThr¹⁷⁵ tau (1:2500; 21st Century #MM-0.147-P), pThr²³¹ tau (1:2500; Thermo Fisher #MN1040), GFP (1:2500; Invitrogen #A-11121), pSer⁹ GSK3β (1:1000; Abcam #ab107166), pRab10 (1:1000; Abcam # ab230621), GSK3β (1:5000; BD Biosciences #610202), or GAPDH (1:4000, Abcam #ab9485) were incubated in blocking solution overnight at 4°C. Membranes were washed in TBST and then incubated at RT in HRP-conjugated goat anti-rabbit (1:3000, Invitrogen #65-6120) or goat anti-mouse (1:3000, Bio-Rad #1706516) for 1 h, washed and signals visualized using chemiluminescent substrate (Perkin Elmer #NEL103E001EA) and a Bio-Rad ChemiDoc XRS+ Gel Imaging System (Bio-Rad). Densitometric analysis was conducted using ImageLab software (Bio-Rad). Membranes were stripped using Restore^™ western blot stripping buffer (Thermo Fisher Scientific #21059) for 20 min at 37°C and 20 min at RT, then washed with TBST and incubated in primary and secondary antibodies as described above for subsequent probing.

For slot blots, the nitrocellulose membrane was pre-soaked in TBS and placed in the Bio-Dot SF Microfiltration Apparatus (Bio-Rad #1706542). The membrane was rehydrated by adding TBS to each well, followed by adding equal amounts of protein diluted to a final volume of 200 μL with lysis buffer. Once the samples had been filtered through the membrane by gentle vacuum, the membrane was rewashed with TBS. The membrane was then incubated in 5% BSA in TBST blocking solution for 1h at room temperature. The primary antibody against LRRK2 (1:1000; Abcam #133474) was incubated overnight at 4°C in blocking solution. Membranes were washed in TBST and incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:3000, Invitrogen #65-6120) for 1h at room temperature. Membranes were washed and visualized as per above with a chemiluminescent substrate.

Statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software, California, United States). Data are represented as mean ± SEM. One-way or two-way ANOVA with Tukey’s and Šidák’s multiple comparisons tests were used for statistical analysis. Statistical significance was defined as p < 0.05.

We first sought to confirm LRRK2 as a candidate kinase for the phosphorylation of Thr¹⁷⁵ tau following TBI. Using immunohistochemistry, we examined the temporal and spatial expression of pThr¹⁷⁵ tau and total LRRK2 in the first 10 days post-injury in rats subject to a single CCI model of TBI. Consistent with our previous data (Hintermayer et al., 2019), pThr¹⁷⁵ tau was present in TBI rodents one-day post-injury with evidence of pThr¹⁷⁵ tau immunoreactive fibrils in the cortex, hippocampus, and corpus callosum detected by day 10 (Figure 1A). Stereotypical tau pathology in the form of dystrophic and corkscrew neurites, and neuropil granules was evident. Non-injured controls displayed consistently low levels of pThr¹⁷⁵ tau (Figure 1A).

Total LRRK2 expression in injured animals was increased in the pericontusional penumbra and hippocampus within the first 10 days compared to non-injured controls (Figure 1B). Specifically, total LRRK2 expression appeared elevated in TBI rodents as early as day 1, peaked at day 5, and persisted to day 10 (Figure 1B). Notably, the greatest DAB intensity (interpreted as the highest level of LRRK2 expression) was detected in cells adjacent to the injury site. This increased expression of LRRK2 was temporally aligned with increased pThr¹⁷⁵ tau following TBI.

In addition to elevated total levels, we evaluated the cellular pattern of neuronal LRRK2 expression. In non-injured controls, LRRK2 expression was predominantly restricted to the cytosol. In contrast, in TBI animals, we observed that the neuronal localization of LRRK2 appeared to be both perikaryal and neuritic, with more pronounced axonal LRRK2 expression. Despite this, we did not observe an increase in the percentage of LRRK2 immunoreactive neurons between control and TBI animals (Figures 2A,B). These data show that increased LRRK2 expression in TBI rodents spatially aligns with increased pThr¹⁷⁵ tau, which further supported the rationale to investigate LRRK2 as a candidate kinase for the phosphorylation of tau at Thr¹⁷⁵.

To further understand the relationship between LRRK2 and pThr¹⁷⁵ tau, and LRRK2 and TBI, we investigated the cellular expression of LRRK2 following TBI. Using immunofluorescence, we examined in which specific cell type LRRK2 expression was increased by double-labelling of LRRK2 and neurons (NeuN), microglia (Iba1), or astrocytes (GFAP). In TBI animals, LRRK2 expression was detected to some extent in all cell types, neurons (Figure 2A), microglia (Figure 2C) and astrocytes (Figure 2F). The most prominent expression of LRRK2 was observed in microglia, with about 80% of Iba1⁺ microglia expressing LRRK2 (Figures 2C,D). The microglia in TBI animals had a stereotypical activated, ameboid morphology associated with inflammation and diseased states. While in general, there was no difference in the percentage of microglia that expressed LRRK2 between TBI and non-injured controls (with the exception of day 5) (Figure 2D), the total number of microglia was significantly increased in TBI compared to controls from day 3 to 10 (Figure 2E). Similarly, while there was no change in the percentage of astrocytes that expressed LRRK2 following TBI (Figure 2G), there was a significant increase in the total number of astrocytes in TBI animals compared to non-injured controls (Figure 2H). The percentage of LRRK2-positive astrocytes was much lower than LRRK2-positive microglia, with only 10%–30% of astrocytes expressing LRRK2. Despite the scarcity of LRRK2 and GFAP co-localization, astrocytes in TBI animals adopted a reactive state and were often observed directly adjacent to LRRK2⁺ cells, with their processes extended. Thus, the increase in total LRRK2 expression in TBI can be attributed to an increase in total number of LRRK2 immunoreactive microglia and astrocytes.

Given that LRRK2 expression is increased within the first 10 days post-injury in TBI rodents, we investigated whether LRRK2 could phosphorylate tau at Thr¹⁷⁵ in vitro. HEK293T cells were co-transfected with eGFP tagged 2N4R human WT-tau and one of three LRRK2 constructs: LRRK2-WT, LRRK2-G2019S (constitutively active) or LRRK2-3XKD (inactive) at 1, 2 and 4 μg. Transfection of WT-tau alone was used as a control. First, we measured the level of LRRK2 expression (Supplementary Figure S1). Given that each LRRK2 construct expressed at differing levels, when measuring pTau and pGSK3β we compared conditions with the most similar level of LRRK2 expression. There was no significant difference in the level of pThr¹⁷⁵ tau in cells overexpressing the constitutively active LRRK2-G2019S compared to LRRK2-WT or the kinase-dead LRRK2-3XKD (Figure 3A; Supplementary Figure S2B). To confirm these data, we co-transfected WT tau and a second kinase dead LRRK2 mutant, LRRK2-K1609M (Gloeckner et al., 2006; Chia et al., 2014). There was no significant difference in the level of pThr¹⁷⁵ tau between LRRK2-G2019S or LRRK2-WT and LRRK2-K1906M (data not shown). Taken together, these results suggest that LRRK2 is not involved in the phosphorylation of Thr¹⁷⁵ of tau.

To confirm our findings, we examined downstream markers of our previously characterized cellular pathway, including pSer⁹-GSK3β (as a marker of GSK3β activity) and pThr²³¹ tau. If LRRK2 is involved in Thr¹⁷⁵ tau phosphorylation, these downstream markers should also be increased. We observed no significant difference in pThr²³¹ tau (Figure 3B) or pSer⁹-GSK3β (Figure 3C) between LRRK2-G2019S and LRRK2-WT or LRRK2-3XKD expressing cells. These data show that LRRK2 does not initiate the cellular cascade associated with pThr¹⁷⁵ tau-induced fibril formation, further supporting that LRRK2 does not phosphorylate Thr¹⁷⁵ of tau.

To validate that the constructs were active, we examined the phosphorylation of a known LRRK2 substrate, Rab10 (Steger et al., 2016). HEK293T cells were co-transfected with eGFP-tagged Rab10 and one of three LRRK2 constructs. The level of pRab10 was significantly greater with the expression of LRRK2-G2019S compared to LRRK2-WT (p = 0.0038) or LRRK2-3XKD (p = 0.0016) (Figure 3D), showing that the findings from the in vitro experiments were not due to inactivity of LRRK2.