Results

Recently, LRRK2 KO rats in the outbred Long–Evans background (Charles Rivers) became commercially available. Zinc-finger nucleases were used to genetically modify the LRRK2 gene with an in-del in exon 30 that results in a premature stop codon in the same exon (Fig. S1A). We verified complete knockdown of protein using both N-terminal and C-terminal targeting LRRK2 monoclonal antibodies (Fig. S1B). We performed stereological analysis in a cohort of rats to estimate the normal content of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the SNpc. We observed no differences, indicating that LRRK2 is not required for dopaminergic neuron development in rats (Fig. S1C).

In vitro evidence suggests that LRRK2 knockdown attenuates proinflammatory responses induced by LPS exposure (10). We first evaluated several endotoxin dosages of ultrapurified LPS (Invivogen, Inc.) in rodents and found that 50 kE.U. (endotoxin units) produced a significant lesion 2 wk after introduction to the SNpc. Because outbred strains like Long–Evans are known for heterogeneity in phenotype and often demand larger groups for appropriately powered analyses, we assembled a cohort of 38 LRRK2 WT and KO rats (n = 19 per genotype) to evaluate the effects of LRRK2 knockdown on dopaminergic neuron survival after LPS exposure. Intracranial injection of LPS into the SNpc of WT rats resulted in an average loss of ∼40% of the population of TH-positive neurons in comparison with the loss of ∼20% observed in an equally sized cohort of LRRK2 knockout rats (Fig. 1 A and B and Fig. S1D). In contrast, introduction of the vehicle control, PBS, into the SNpc failed to cause any significant loss of TH-positive neurons in either LRRK2 WT or KO rats (Fig. S1E).

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Fig. 1.

Absence of LRRK2 protects from LPS-induced dopaminergic neurodegeneration. LRRK2 WT or KO rats, aged 10–12 wk, were unilaterally injected with either 50 kE.U. (from 10 μg of LPS) or vehicle (PBS) control, and killed 2 wk postinjection. (A) Representative coronal sections of TH-stained SNpc and (B) unbiased stereological counts of TH-positive neurons in the SNpc, with injected side (ipsilateral) counts and noninjected side (contralateral) counts given. Counts expressed as a percentage TH-positive cell loss are shown in Fig. S1 D and E. (C) Stereological counts of CD68-positive myeloid cells in the ipsilateral midbrain sections. Contralateral midbrain sections have negligible CD68 immunoreactivity (<100 cells) and counts are not shown. (D) Representative coronal sections showing CD68 immunoreactivity in the SNpc injected with LPS. (E) Correlation between recruitment of CD68-positive myeloid cells and percentage of surviving TH cells in the SNpc. (F) Perimeter analysis of cells labeled with Iba1 through the midbrain (n = 3 animals per group, >1,000 Iba1 cells analyzed per group). (G) Representative confocal images that span the ipsilateral (LPS-exposed) SNpc. [Scale bar, 0.5 mm for A and D and 100 μm (5 μm for high-magnification insets) for G.] P values are calculated by one-way ANOVA with Tukey’s post hoc test (B, C, D, and F), and linear regression analysis for E (Pearson’s r = −0.75). Error bars represent SEM.

LRRK2 expression has been detected in activated myeloid cells in the periphery and brain (10, 11). We used CD68, a glycoprotein expressed in cells of the myeloid lineage, as a marker for activated microglia, macrophages, and monocytes. Although few or no CD68-positive cells could be detected in the noninjected SNpc in rats (always <100 cells across the whole SNpc in the cohort; Fig. 1D), LPS exposure induced a significant recruitment of CD68-positive cells in the injected SNpc (Fig. 1 C and D). Vehicle control injection alone resulted in only modest CD68-positive cell induction (Fig. 1C). In rats lacking LRRK2, stereological counts demonstrated a significant reduction in CD68-positive cell recruitment compared with WT rats (Fig. 1C). The number of CD68-positive cells recruited to the midbrain correlated with dopaminergic cell death in the SNpc (Fig. 1E). A shift from ramified to amoeboid morphologies of ionized calcium-binding adapter molecule 1 (Iba1)/CD68-positive cells is indicative of myeloid cell activation (15). In LPS-exposed tissue, all CD68-positive cells were also Iba1-positive, and analysis of cell contours illuminated by Iba1 staining in the midbrain demonstrated a shift from ramified to amoeboid cells in LPS- versus PBS-exposed SNpc tissue in WT rats. LRRK2 KO rats failed to develop comparable amoeboid cell morphologies despite robust CD68 expression (Fig. 1F). Inducible nitric oxide synthase (iNOS) is a classical proinflammatory marker and could be detected in all CD68-positive cells in both LRRK2 WT and KO myeloid cells after LPS exposure. However, levels of iNOS were much more prominent in CD68-positive cells in the WT rats compared with the LRRK2 KO rats (Fig. 1G). These results demonstrate that ablation of LRRK2 expression significantly reduced both dopaminergic neurodegeneration induced by LPS exposure as well as CD68-positive myeloid cell recruitment.

LPS has been applied to both in vitro and in vivo paradigms to investigate myeloid cell-derived neuroinflammatory responses. Direct α-synuclein exposures to microglia cells have been described to activate many of the same responses (16). α-synuclein overexpression in the SNpc using recombinant adeno-associated virus (rAAV) has been previously found to produce a selective and relatively robust lesion of dopaminergic neurons, comparable to the level of neurodegeneration induced by LPS exposure (12, 13). We titrated the rAAV virus expressing human WT α-synuclein to determine the minimum amount required to reliably transduce >90% of dopaminergic neurons in the SNpc of Long–Evans LRRK2 WT and KO rats, as well as to ensure that LRRK2 WT and KO rats were similarly susceptible to viral transduction. We selected the amount of 6 × 10⁹ viral particles delivered to the SNpc, and no differences were noted in the efficiency of transduction in LRRK2 WT or KO rats (Fig. S2 B and C). Next, we measured the expression of exogenous and endogenous α-synuclein in both LRRK2 WT and KO rat midbrains 2 wk after intracranial injections. We found comparable expression levels of total α-synuclein levels as well as comparable soluble and insoluble α-synuclein levels (Fig. S3 A–C).

To determine whether LRRK2 modifies α-synuclein–induced dopaminergic neurodegeneration, α-synuclein or eGFP viruses were unilaterally injected in 10–12-wk-old LRRK2 WT and KO rats (n = 100). Animals were killed at 4 wk, 8 wk, and 12 wk postinjection. Four animals, three KO and one WT, were excluded from this study due to an incomplete viral transduction of the SNpc and/or unsuccessful tissue processing. α-synuclein expression was evaluated in tissue sections using a human-isoform–specific monoclonal antibody (Syn208) or an antibody selective for α-synuclein pathologic inclusions (Syn514). In SNpc neurons in WT or KO rats labeled by either antibody, intracellular distribution of α-synuclein showed comparable punctate cytoplasmic staining (Fig. 2 B and C). Surprisingly, LRRK2 KO animals showed more robust abnormal α-synuclein reactivity across the SNpc (Fig. 2A).

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Fig. 2.

Absence of LRRK2 protects from α-synuclein–induced dopaminergic neurodegeneration. Analysis of 10–12-wk-old LRRK2 WT and KO rats unilaterally injected with 6 × 10⁹ rAAV2 α-synuclein viral particles. (A) Representative DAB images with Nissl counterstain showing human α-synuclein immunoreactivity in neurons of the ipsilateral SNpc 4 wk after viral injection. (B) Representative high-magnification SNpc neurons showing human α-synuclein immunoreactivity and (C) reactivity with a α-synuclein antibody that selective recognizes pathologic inclusions (23). SNpc sections were analyzed for α-synuclein aggregates in Nissl-positive cells at high magnification. (D) Representative coronal sections showing TH immunoreactivity in the SNpc, 4 wk after viral injection. (E) Unbiased stereological analysis of TH-positive neurons in the SNpc with respect to ipsilateral (ipsi, injected) and contralateral (contra, uninjected) sides. Stereological counts of Nissl-positive cells and dopaminergic neurodegeneration, calculated as percentage of remaining neurons, are provided in Fig. S4. (F) Linear regression analysis for SNpc neurodegeneration, determined by stereological counts of TH cells in the SNpc, and the percentage of cells that remain with α-synuclein aggregates. Additional representative images are provided in Fig. S5. (G) Representative coronal sections showing CD68 immunoreactivity in the SNpc and (H) unbiased stereological analysis of CD68 cells, 4 wk postinjection of rAAV2 α-synuclein viral particles. (I) Perimeter analysis of cells labeled with Iba1 through the midbrain (n = 3 animals per group, >1,000 Iba1 cells analyzed per group). (J) Representative confocal sections that span the ipsilateral (α-synuclein transduced) SNpc. CD68 immunoreactivity was never detected on the contralateral side. [Scale bar, 0.5 mm for A, D, and G and 50 μm (and 5 μm for the high-magnification inset) for J.] P values were calculated by one-way ANOVA with Tukey’s post hoc test, and error bars represent SEM.

Gross losses of SNpc neurons were apparent in WT but not LRRK2 KO rats (Fig. 2D). An overall average loss of ∼30% of TH-positive dopaminergic neurons in WT rats was seen across the 4, 8, and 12 wk post–α-synuclein virus injection (Fig. 2E and Fig. S4 A and B). The extent of neurodegeneration did not vary between the three time points (one-way ANOVA, P > 0.1), indicating a nonprogressive loss of cells. At each of these time points, no significant loss of TH-positive neurons could be detected in LRRK2 KO rats. To verify that the reduction of TH-positive neurons in α-synuclein–transduced WT rats occurred due to cell loss instead of loss of TH expression, Nissl-positive cells in the SNpc were also counted, and similar differences were observed in WT but not KO animals (Fig. S4A). As with the LPS-injected rat cohorts, no significant sex effects were observed in any group. The effects of matched titers of EGFP control virus did not vary between the LRRK2 WT and KO animals (Fig. S4C). Prominent α-synuclein pathology could be detected in SNpc in both WT and LRRK2 KO rats. We observed a significant (P < 0.003) inverse correlation between α-synuclein pathology and the extent of neurodegeneration (Fig. 2F). In rats with severe loss of TH-positive SNpc cells (e.g., 40%), fewer neurons were detected with abnormal α-synuclein expression (Fig. S5). Because viral transduction and α-synuclein expression is equivalent at earlier time points before cell loss (Figs. S2 and S3), transduced SNpc neurons with abnormal α-synuclein were lost in WT but not LRRK2 KO rats.

Microglial activation has been previously described in rAAV2–α-synuclein–transduced rodents (12, 14, 17). In WT rats transduced with α-synuclein, we observed a significant recruitment of CD68-positive cells within the SNpc (Fig. 2 G–J). Consistent with observations made in rats with LPS exposures (Fig. 1), the number of CD68-positive cells recruited in response to α-synuclein expression were reduced in LRRK2 KO rats compared with WT rats (Fig. 2H). In addition, there was a significant shift from ramified to amoeboid Iba1-positive cells in WT rats (Fig. 2 I and J). These amoeboid, CD68/Iba1-positive cells that accumulated in the WT, but not LRRK2 KO, rats also demonstrated higher iNOS expression than ramified cells (Fig. 2J). To probe LRRK2 expression within the myeloid cell fields induced by α-synuclein expression, we costained sections using LRRK2 monoclonal antibodies with optimized immunohistochemical protocols (18) in both ipsilateral and contralateral SNpc (Fig. 3). LRRK2 signal overlapped well with CD68-positive cells. Colabeling experiments demonstrated that the highest LRRK2-expressing myeloid cells were also the strongest iNOS-expressing cells (Fig. 3 A–D). In contrast, LRRK2 was not or weakly expressed in noninjected SNpc cells (Fig. 3E). Similarly, LPS-induced Iba1/iNOS-positive cells also expressed LRRK2 protein (Fig. 3 I–L). Thus, in WT rats, proinflammatory myeloid cells were the only LRRK2-positive cells that were detected in the midbrain. In LRRK2 KO rats, these proinflammatory myeloid cells were reduced in number and had reduced iNOS expression.

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Fig. 3.

LRRK2 immunoreactivity in CD68/iNOS-positive cells recruited in response to α-synuclein and LPS-transduced SNpc. (A–D) Representative confocal sections of the unilateral SNpc transduced with rAAV2/1–α-synuclein, into 10–12-wk-old LRRK2 WT rats, killed 4 wk postinjection. LRRK2 (green)/CD68 (red)/iNOS (blue)-positive small amoeboid (spherical) cells (e.g., ∼5 μm) are highlighted with arrowheads. (E–H) In the contralateral side (noninjected), no immunoreactivity could be detected for LRRK2, CD68, and iNOS. (I–L) LRRK2 WT rats, aged 10–12 wk, were unilaterally injected with 50 kE.U. (from 10 μg of LPS) and killed 2 wk postinjection. Confocal analysis of a representative area of the ipsilateral SNpc revealed cells positive for LRRK2, Iba1, CD68, and iNOS. Arrowheads indicate points of reference. (Scale bar, 50 μm for A–H and 20 μm for I–L.)