Etsuro Ohta

An LRRK2 mutation as a cause for the parkinsonism in the original PARK8 family

We detected a missense mutation in the kinase domain of the LRRK2 gene in members with autosomal dominant Parkinsons disease of the Japanese family (the Sagamihara family) who served as the basis for the original defining of the PARK8 Parkinsons disease locus. The results of the Sagamihara family, in combination with the unique pathological features characterized by pure nigral degeneration without Lewy bodies, provided us with valuable information for elucidating the protein structure–pathogenesis relationship for the gene product of LRRK2. We did not detect this mutation or other known mutations of the LRRK2 gene in Japanese patients with sporadic Parkinsons disease. Ann Neurol 2005

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 Parkinsons 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.

LRRK2 directly phosphorylates Akt1 as a possible physiological substrate: impairment of the kinase activity by Parkinson's disease-associated mutations.

LRRK2 binds to Akt1 by pull down (View Interaction 1, 2, 3).

Stimulatory effect of α-synuclein on the tau-phosphorylation by GSK-3β

Hyperphosphorylation of tau protein (tau) causes neurodegenerative diseases such as Alzheimer’s disease (AD). Recent studies of the physiological correlation between tau and α‐synuclein (α‐SN) have demonstrated that: (a) phosphorylated tau is also present in Lewy bodies, which are cytoplasmic inclusions formed by abnormal aggregation of α‐SN; and (b) the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) increases the phosphorylation of tau as well as the protein level of α‐SN in cultured neuronal cells, and also in mice. However, the molecular mechanism responsible for the α‐SN‐mediated hyperphosphorylation of tau remains to be elucidated. In this in vitro study, we found that: (a) α‐SN directly stimulates the phosphorylation of tau by glycogen synthase kinase‐3β (GSK‐3β), (b) α‐SN forms a heterotrimeric complex with tau and GSK‐3β, and (c) the nonamyloid beta component (NAC) domain and an acidic region of α‐SN are responsible for the stimulation of GSK‐3β‐mediated tau phosphorylation. Thus, it is concluded that α‐SN functions as a connecting mediator for tau and GSK‐3β, resulting in GSK‐3β‐mediated tau phosphorylation. Because the expression of α‐SN is promoted by oxidative stress, the accumulation of α‐SN induced by such stress may directly induce the hyperphosphorylation of tau by GSK‐3β. Furthermore, we found that heat shock protein 70 (Hsp70) suppresses the α‐SN‐induced phosphorylation of tau by GSK‐3β through its direct binding to α‐SN, suggesting that Hsp70 acts as a physiological suppressor of α‐SN‐mediated tau hyperphosphorylation. These results suggest that the cellular level of Hsp70 may be a novel therapeutic target to counteract α‐SN‐mediated tau phosphorylation in the initial stage of neurodegenerative disease.

Age-dependent and cell-population-restricted LRRK2 expression in normal mouse spleen.

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinsons disease (PD), but its true physiological function remains unknown. In the normal mouse, LRRK2 is expressed in kidney, spleen, and lung at much higher levels than in brain, suggesting that LRRK2 may play an important role in these organs. Analysis of age-related changes in LRRK2 expression demonstrated that expression in kidney, lung, and various brain regions was constant throughout adult life. On the other hand, expression of both LRRK2 mRNA and protein decreased markedly in spleen in an age-dependent manner. Analysis of purified spleen cells indicated that B lymphocytes were the major population expressing LRRK2, and that T lymphocytes showed no expression. Consistently, the B lymphocyte surface marker CD19 exhibited an age-dependent decrease of mRNA expression in spleen. These results suggest a possibly novel function of LRRK2 in the immune system, especially in B lymphocytes.

Leucine-rich repeat kinase 2 regulates tau phosphorylation through direct activation of glycogen synthase kinase-3β.

Leucine‐rich repeat kinase 2 (LRRK2) has been identified as the causal molecule for autosomal‐dominant Parkinsons disease (PD). Experimental evidence indicates that LRRK2 may play an important role in the pathology induced by abnormal phosphorylation of tau. In the present study, we demonstrated that LRRK2 directly associates with GSK‐3β, and that this interaction enhances the kinase activity of GSK‐3β. Furthermore, we found that LRRK2‐mediated activation of GSK‐3β induces high phosphorylation of tau at Ser396 in SH‐SY5Y cells. From our present findings, we conclude that LRRK2 may function as a novel enhancer for GSK‐3β and as a physiological regulator of neurite outgrowth and axonal transport through regulation of the GSK‐3β‐mediated phosphorylation of tau at the cellular level. Since LRRK2 is detected in tau‐positive inclusions in brain tissue affected by various neurodegenerative disorders, including PD, LRRK2‐stimulated phosphorylation of tau by GSK‐3β may be involved in development of pathological features in the initial stage of PD.

LRRK2 is expressed in B-2 but not in B-1 B cells, and downregulated by cellular activation

LRRK2, the causal molecule of familial Parkinsons disease, is expressed strongly by one of the B cell subsets, B-2 cells, but not by the other subset, B-1 cells, in the mouse peritoneal cavity, spleen, and peripheral blood. Bone marrow pre-B cells or T cells exhibited little LRRK2 expression. LRRK2 expression was dramatically downregulated upon activation of B-2 cells with various types of stimulation. These results suggest that LRRK2, whose true function has not yet been clarified, may play some important role(s) in the development and function of B cells, particularly the maintenance of B-2 cells in a resting status.

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

BackgroundLeucine-rich repeat kinase 2 (LRRK2) 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.ResultsThe 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.ConclusionsThe 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.

I2020T mutant LRRK2 iPSC-derived neurons in the Sagamihara family exhibit increased Tau phosphorylation through the AKT/GSK-3β signaling pathway

Leucine-rich repeat kinase 2 (LRRK2) is the causative molecule of the autosomal dominant hereditary form of Parkinsons disease (PD), PARK8, which was originally defined in a study of a Japanese family (the Sagamihara family) harboring the I2020T mutation in the kinase domain. Although a number of reported studies have focused on cell death mediated by mutant LRRK2, details of the pathogenetic effect of LRRK2 still remain to be elucidated. In the present study, to elucidate the mechanism of neurodegeneration in PD caused by LRRK2, we generated induced pluripotent stem cells (iPSC) derived from fibroblasts of PD patients with I2020T LRRK2 in the Sagamihara family. We found that I2020T mutant LRRK2 iPSC-derived neurons released less dopamine than control-iPSC-derived neurons. Furthermore, we demonstrated that patient iPSC-derived neurons had a lower phospho-AKT level than control-iPSC-derived neurons, and that the former showed an increased incidence of apoptosis relative to the controls. Interestingly, patient iPSC-derived neurons exhibited activation of glycogen synthase kinase-3β (GSK-3β) and high Tau phosphorylation. In addition, the postmortem brain of the patient from whom the iPSC had been established exhibited deposition of neurofibrillary tangles as well as increased Tau phosphorylation in neurons. These results suggest that I2020T LRRK2-iPSC could be a promising new tool for reproducing the pathology of PD in the brain caused by the I2020T mutation, and applicable as a model in studies of targeted therapeutics.

I2020T leucine-rich repeat kinase 2, the causative mutant molecule of familial Parkinson’s disease, has a higher intracellular degradation rate than the wild-type molecule

Leucine-rich repeat kinase 2 (LRRK2) has been identified as the causal gene for autosomal dominant familial Parkinsons disease (PD), although the mechanism of neurodegeneration involving the mutant LRRK2 molecules remains unknown. In the present study, we found that the protein level of transfected I(2020)T mutant LRRK2 was significantly lower than that of wild-type and G(2019)S mutant LRRK2, although the intracellular localization of the I(2020)T and wild-type molecules did not differ. Pulse-chase experiments proved that the I(2020)T LRRK2 molecule has a higher degradation rate than wild-type or G(2019)S LRRK2. Upon addition of proteasome and lysosome inhibitors, the protein level of I(2020)T mutant LRRK2 reached that of the wild-type. These results indicate that I(2020)T mutant LRRK2 is more susceptible to post-translational degradation than the wild-type molecule. Our results indicate a novel molecular feature characteristic to I(2020)T LRRK2, and provide a new insight into the mechanism of neurodegeneration caused by LRRK2.