LRRK2 Phosphorylates Tubulin-Associated Tau but Not the Free Molecule: LRRK2-Mediated Regulation of the Tau-Tubulin Association and Neurite Outgrowth

Leucine-rich repeat kinase 2 (LRRK2), a large protein kinase containing multi-functional domains, has been identified as the causal molecule for autosomal-dominant Parkinson's disease (PD). In the present study, we demonstrated for the first time that (i) LRRK2 interacts with tau in a tubulin-dependent manner; (ii) LRRK2 directly phosphorylates tubulin-associated tau, but not free tau; (iii) LRRK2 phosphorylates tau at Thr181 as one of the target sites; and (iv) The PD-associated LRRK2 mutations, G2019S and I2020T, elevated the degree of tau-phosphorylation. These results provide direct proof that tau is a physiological substrate for LRRK2. Furthermore, we revealed that LRRK2-mediated phosphorylation of tau reduces its tubulin-binding ability. Our results suggest that LRRK2 plays an important role as a physiological regulator for phosphorylation-mediated dissociation of tau from microtubules, which is an integral aspect of microtubule dynamics essential for neurite outgrowth and axonal transport

The I2020T Leucine-rich repeat kinase 2 transgenic mouse exhibits impaired locomotive ability accompanied by dopaminergic neuron abnormalities

<p>Abstract</p> <p>Background</p> <p><it>Leucine-rich repeat kinase 2 (LRRK2)</it> is the gene responsible for autosomal-dominant Parkinson’s disease (PD), PARK8, but the mechanism by which LRRK2 mutations cause neuronal dysfunction remains unknown. In the present study, we investigated for the first time a transgenic (TG) mouse strain expressing human LRRK2 with an I2020T mutation in the kinase domain, which had been detected in the patients of the original PARK8 family.</p> <p>Results</p> <p>The TG mouse expressed I2020T LRRK2 in dopaminergic (DA) neurons of the substantia nigra, ventral tegmental area, and olfactory bulb. In both the beam test and rotarod test, the TG mice exhibited impaired locomotive ability in comparison with their non-transgenic (NTG) littermates. Although there was no obvious loss of DA neurons in either the substantia nigra or striatum, the TG brain showed several neurological abnormalities such as a reduced striatal dopamine content, fragmentation of the Golgi apparatus in DA neurons, and an increased degree of microtubule polymerization. Furthermore, the tyrosine hydroxylase-positive primary neurons derived from the TG mouse showed an increased frequency of apoptosis and had neurites with fewer branches and decreased outgrowth in comparison with those derived from the NTG controls.</p> <p>Conclusions</p> <p>The I2020T LRRK2 TG mouse exhibited impaired locomotive ability accompanied by several dopaminergic neuron abnormalities. The TG mouse should provide valuable clues to the etiology of PD caused by the LRRK2 mutation.</p

Leucine-rich repeat kinase 2 (LRRK2) regulates α-synuclein clearance in microglia

Background: α-Synuclein (αSYN) has been genetically implicated in familial and sporadic Parkinson’s disease (PD), and is associated with disease susceptibility, progression and pathology. Excess amounts of αSYN are toxic to neurons. In the brain, microglial αSYN clearance is closely related to neuronal survival. Leucine-rich repeat kinase 2 (LRRK2) is the one of the other genes implicated in familial and sporadic PD. While LRRK2 is known to be expressed in microglia, its true function remains to be elucidated. In this study, we investigated αSYN clearance by microglia isolated from LRRK2-knockout (KO) mice. Results: In LRRK2-KO microglia, αSYN was taken up in larger amounts and cleared from the supernatant more effectively than for microglia isolated from wild-type (WT) mice. This higher clearance ability of LRRK2-KO microglia was thought to be due to an increase of Rab5-positive endosomes, but not Rab7- or Rab11-positive endosomes. Increased engagement between Rab5 and dynamin 1 was also observed in LRRK2-KO microglia. Conclusion: LRRK2 negatively regulates the clearance of αSYN accompanied by down-regulation of the endocytosis pathway. Our findings reveal a new functional role of LRRK2 in microglia and offer a new insight into the mechanism of PD pathogenesis.Medicine, Faculty ofOther UBCNon UBCMedical Genetics, Department ofReviewedFacult

Leucine-Rich Repeat Kinase 2 Controls Inflammatory Cytokines Production through NF-κB Phosphorylation and Antigen Presentation in Bone Marrow-Derived Dendritic Cells

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinson&rsquo;s disease. Although the characteristics of LRRK2 have gradually been revealed, its true physiological functions remain unknown. LRRK2 is highly expressed in immune cells such as B2 cells and macrophages, suggesting that it plays important roles in the immune system. In the present study, we investigate the roles of LRRK2 in the immune functions of dendritic cells (DCs). Bone marrow-derived DCs from both C57BL/6 wild-type (WT) and LRRK2 knockout (KO) mice were induced by culture with granulocyte/macrophage-colony stimulating factor (GM/CSF) in vitro. We observed the differentiation of DCs, the phosphorylation of the transcriptional factors NF-&kappa;B, Erk1/2, and p-38 after lipopolysaccharide (LPS) stimulation and antigen-presenting ability by flow cytometry. We also analyzed the production of inflammatory cytokines by ELISA. During the observation period, there was no difference in DC differentiation between WT and LRRK2-KO mice. After LPS stimulation, phosphorylation of NF-&kappa;B was significantly increased in DCs from the KO mice. Large amounts of inflammatory cytokines were produced by DCs from KO mice after both stimulation with LPS and infection with Leishmania. CD4+ T-cells isolated from antigen-immunized mice proliferated to a significantly greater degree upon coculture with antigen-stimulated DCs from KO mice than upon coculture with DCs from WT mice. These results suggest that LRRK2 may play important roles in signal transduction and antigen presentation by DCs

Cardiac sarcoidosis treated with nonsteroidal immunosuppressive therapy

Background: Nonsteroidal immunosuppressive therapy is a potential therapeutic strategy for cardiac sarcoidosis. However, it is not recommended as an established treatment option. This study aimed to demonstrate the clinical outcomes of patients with cardiac sarcoidosis using nonsteroidal immunosuppressants through the ILLUstration of the Management and PrognosIs of JapaNese PATiEnts with Cardiac Sarcoidosis multicenter retrospective registry. Methods: From a cohort of 512 patients, 426 who received corticosteroid therapy and 26 who received other immunosuppressive therapy were included for analysis. Clinical outcomes included all-cause death, fatal ventricular arrhythmic events (FVAE), and worsening heart failure with hospitalization. Results: Nonsteroidal immunosuppressants were used for retained fluorodeoxyglucose uptake in the heart (n = 14), corticosteroid side effects (n = 7), ventricular arrhythmia (n = 4), complete atrioventricular block (n = 2), worsened extracardiac sarcoidosis (n = 2), and other reasons (n = 2). They comprised of methotrexate (n = 20), cyclosporine (n = 2), cyclophosphamide (n = 2), and azathioprine (n = 3). After the addition of a nonsteroidal immunosuppressant, corticosteroids were reduced in 14 of 26 patients (5 [5–17] mg), although no patient discontinued corticosteroids. Of the 14 patients, decreased fluorodeoxyglucose uptake was observed in seven at follow-up. Clinical outcomes were observed in 11 patients (42.3 %). Detected events included all-cause death in five patients (19.2 %), FVAE in four (15.4 %), and worsening heart failure with hospitalization in five (19.2 %), with some overlap. Conclusions: Nonsteroidal immunosuppressive therapy may be a possible treatment option for patients who are not stabilized with corticosteroids alone or develop corticosteroid side effects

LRRK2-mediated regulation of association/dissociation between tau and tubulin.

<p>LRRK2 interacts with tubulin-associated tau, resulting in the formation of a tripartite complex (lower left). This complex induces the phosphorylation of tau by LRRK2 (upper left) and sequentially induces dissociation of tau from tubulin (upper right). Tau is dephosphorylated by certain protein phosphatases, and the dephosphorylated tau then recovers its ability to bind with tubulin (lower right).</p

Tubulin-dependent interaction of LRRK2 with tau.

<p>(A) V5-LRRK2 was immunoprecipitated with anti-V5-agarose beads from the V5-LRRK2-stably-expressing SH-SY5Y clone, WT4-D33. Normal rabbit IgG-conjugated agarose beads were used as a control. The immunoprecipitates were separated by SDS-PAGE, transferred to PVDF, and analyzed using antibodies against V5-tag, tau, and tubulin. (B) GST (lane 1) and GST-LRRK2 (lane 2) were incubated with porcine tubulin, and a pull-down assay using glutathione-agarose beads was performed. Precipitated proteins were detected by Western analysis using anti-GST and anti-tubulin antibodies. (C) GST and GST-LRRK2 were incubated with recombinant tau in the absence (lane 1 and 2) or presence (lane 3 and 4) of porcine tubulin, and a pull-down assay using glutathione-agarose beads was performed. Precipitated proteins were detected by Western analysis using the indicated antibodies.</p

Effect of LRRK2-mediated phosphorylation on the ability of tau to bind to tubulin.

<p>(A) Recombinant tau (2 µg) was incubated with GST-LRRK2 (50 ng) and 3 µCi of [γ-³²P]ATP in the presence of porcine tubulin (1 µg) at 30°C for 120 min and then a GST pull-down assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030834#s2" target="_blank">Materials and Methods</a>. ³²P-Labeled proteins in the precipitates were detected by autoradiography following SDS-PAGE (upper panel). Total tau in the same sample was detected by Western blotting analysis using anti-tau antibody (lower panel). (B) V5-LRRK2 was immunoprecipitated with anti-V5-agarose beads from a WT4-D33 cell lysate. Precipitated proteins were analyzed by Western blotting using antibodies against V5-tag, phospho-tau (Thr181), non-phospho-epitopes of tau, and tubulin.</p