Biphenotypic Cells and α-Synuclein Accumulation in Enteric Neurons of Leucine-Rich Repeat Kinase 2 Knockout Mice.

BACKGROUND: Leucine-rich repeat kinase 2 is a molecule that is responsible for familial Parkinson's disease. Our previous findings revealed that leucine-rich repeat kinase 2 is expressed in the enteric nervous system. However, which cells in the enteric nervous system express leucine-rich repeat kinase 2 and whether leucine-rich repeat kinase 2 is associated with the structure of the enteric nervous system remain unclear. The enteric nervous system is remarkable because some patients with Parkinson's disease experience gastrointestinal symptoms before developing motor symptoms. AIMS: We established a leucine-rich repeat kinase 2 reporter mouse model and performed immunostaining in leucine-rich repeat kinase 2 knockout mice. METHODS: Longitudinal muscle containing the myenteric plexus prepared from leucine-rich repeat kinase 2 reporter mice was analyzed by immunostaining using anti-green fluorescent protein (GFP) antibody. Immunostaining using several combinations of antibodies characterizing enteric neurons and glial cells was performed on intestinal preparations from leucine-rich repeat kinase 2 knockout mice. RESULTS: GFP expression in the reporter mice was predominantly in enteric glial cells rather than in enteric neurons. Immunostaining revealed that differences in the structure and proportion of major immunophenotypic cells were not apparent in the knockout mice. Interestingly, the number of biphenotypic cells expressing the neuronal and glial cell markers increased in the leucine-rich repeat kinase 2 knockout mice. Moreover, there was accumulation of α-synuclein in the knockout mice. CONCLUSIONS: Our present findings suggest that leucine-rich repeat kinase 2 is a newly recognized molecule that potentially regulates the integrity of enteric nervous system and enteric α-synuclein accumulation.

LRRK2 negatively regulates glucose tolerance via regulation of membrane translocation of GLUT4 in adipocytes.

Epidemiological studies have shown that abnormalities of glucose metabolism are involved in leucine-rich repeat kinase 2 (LRRK2)-associated Parkinson's disease (PD). However, the physiological significance of this association is unclear. In the present study, we investigated the effect of LRRK2 on high-fat diet (HFD)-induced glucose intolerance using Lrrk2-knockout (KO) mice. We found for the first time that HFD-fed KO mice display improved glucose tolerance compared with their wild-type (WT) counterparts. In addition, high serum insulin and leptin, as well as low serum adiponectin resulting from HFD in WT mice were improved in KO mice. Using western blotting, we found that Lrrk2 is highly expressed in adipose tissues compared with other insulin-related tissues that are thought to be important in glucose tolerance, including skeletal muscle, liver, and pancreas. Lrrk2 expression and phosphorylation of its kinase substrates Rab8a and Rab10 were significantly elevated after HFD treatment in WT mice. In cell culture experiments, treatment with a LRRK2 kinase inhibitor stimulated insulin-dependent membrane translocation of glucose transporter 4 (Glut4) and glucose uptake in mouse 3T3-L1 adipocytes. We conclude that increased LRRK2 kinase activity in adipose tissue exacerbates glucose tolerance by suppressing Rab8- and Rab10-mediated GLUT4 membrane translocation.

Interleukin-13 Mediates Non-Steroidal Anti-Inflammatory-Drug-Induced Small Intestinal Mucosal Injury with Ulceration.

Non-steroidal anti-inflammatory drugs (NSAIDs), which are antipyretics and analgesics, cause gastrointestinal disorders, such as inflammation and ulcers. To prescribe NSAIDs more safely, it is important to clarify the mechanism of NSAID-induced gastrointestinal mucosal injury. However, there is a paucity of studies on small intestinal mucosal damage by NSAIDs, and it is currently unknown whether inflammation and ulceration also occur in the small intestine, and whether mediators are involved in the mechanism of injury. Therefore, in this study, we created an animal model in which small intestinal mucosal injury was induced using NSAIDs (indomethacin; IDM). Focusing on the dynamics of immune regulatory factors related to the injury, we aimed to elucidate the pathophysiological mechanism involved. We analyzed the pathological changes in the small intestine, the expression of immunoregulatory factors (cytokines), and identified cytokine secretion and expression cells from isolated lamina propria mononuclear cells (LPMCs). Ulcers were formed in the small intestine by administering IDM. Although the mRNA expression levels of IL-1ß, IL-6, and TNFα were decreased on day 7 after IDM administration, IL-13 mRNA levels increased from day 3 after IDM administration and remained high even on day 7. The IL-13 mRNA expression and the secretion of IL-13 were increased in small intestinal LPMCs isolated from the IDM-treated group. In addition, we confirmed that IL-13 was expressed in CD4-positive T cells. These results provided new evidence that IL-13 production from CD4-positive T cells in the lamina propria of the small intestine contributes to NSAID-induced mucosal injury.

Nrf2 Expression Is Decreased in LRRK2 Transgenic Mouse Brain and LRRK2 Overexpressing SH-SY5Y Cells.

Mutations in leucine rich-repeat kinase 2 (LRRK2) cause autosomal-dominant, late-onset Parkinson's disease (PD). Accumulating evidence indicates that PD-associated LRRK2 mutations induce neuronal cell death by increasing cellular reactive oxygen species levels. However, the mechanism of increased oxidative stress associated with LRRK2 kinase activity remains unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that protects cells from oxidative stress by inducing the expression of antioxidant genes. In the present, it was found that decreased expression of Nrf2 and mRNA expression of its target genes in Lrrk2-transgenic mouse brain and LRRK2 overexpressing SH-SY5Y cells. Furthermore, knockdown of glycogen synthase kinase-3ß (GSK-3ß) recovered Nrf2 expression and mRNA expression of its target genes in LRRK2 overexpressing SH-SY5Y cells. We concluded that since Nrf2 is transcriptional factor for antioxidative responses, therefore, reduction of Nrf2 expression by LRRK2 may be part of a mechanism that LRRK2-induces vulnerability to oxidative stress in neuronal cells.

LRRK2 Inhibition Ameliorates Dexamethasone-Induced Glucose Intolerance via Prevents Impairment in GLUT4 Membrane Translocation in Adipocytes.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson's disease. LRRK2 is a large protein with multiple functional domains, including a guanosine 5'-triphosphate (GTP)-binding domain and a protein kinase domain. Recent studies indicated that the members of the Rab GTPase family, Rab8a and Rab10, which are involved in the membrane transport of the glucose transporter type 4 (GLUT4) during insulin-dependent glucose uptake, are phosphorylated by LRRK2. However, the physiological role of LRRK2 in the regulation of glucose metabolism is largely unknown. In the present study, we investigated the role of LRRK2 using dexamethasone (DEX)-induced glucose intolerance in mice. LRRK2 knockout (KO) mice exhibited suppressed glucose intolerance, even after treatment with DEX. The phosphorylation of LRRK2, Rab8a and Rab10 was increased in the adipose tissues of DEX-treated wild-type mice. In addition, inhibition of the LRRK2 kinase activity prevented the DEX-induced inhibition of GLUT4 membrane translocation and glucose uptake in cultured 3T3-L1 adipocytes. These results suggest that LRRK2 plays an important role in glucose metabolism in adipose tissues.

Histamine H2-Receptor Antagonists Improve Non-Steroidal Anti-Inflammatory Drug-Induced Intestinal Dysbiosis.

Dysbiosis, an imbalance of intestinal flora, can cause serious conditions such as obesity, cancer, and psychoneurological disorders. One cause of dysbiosis is inflammation. Ulcerative enteritis is a side effect of non-steroidal anti-inflammatory drugs (NSAIDs). To counteract this side effect, we proposed the concurrent use of histamine H2 receptor antagonists (H2RA), and we examined the effect on the intestinal flora. We generated a murine model of NSAID-induced intestinal mucosal injury, and we administered oral H2RA to the mice. We collected stool samples, compared the composition of intestinal flora using terminal restriction fragment length polymorphism, and performed organic acid analysis using high-performance liquid chromatography. The intestinal flora analysis revealed that NSAID [indomethacin (IDM)] administration increased Erysipelotrichaceae and decreased Clostridiales but that both had improved with the concurrent administration of H2RA. Fecal levels of acetic, propionic, and n-butyric acids increased with IDM administration and decreased with the concurrent administration of H2RA. Although in NSAID-induced gastroenteritis the proportion of intestinal microorganisms changes, leading to the deterioration of the intestinal environment, concurrent administration of H2RA can normalize the intestinal flora.

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'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-κB, 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-κB 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.

Leucine-Rich Repeat Kinase 2 Is Associated With Activation of the Paraventricular Nucleus of the Hypothalamus and Stress-Related Gastrointestinal Dysmotility.

Leucine-rich repeat kinase 2 (LRRK2) is a molecule associated with familial and sporadic Parkinson's disease. It regulates many central neuronal functions, such as cell proliferation, apoptosis, autophagy, and axonal extension. Recently, it has been revealed that LRRK2 is related to anxiety/depression-like behavior, implying an association between LRRK2 and stress. In the present study, we investigated for the first time the stress pathway and its relationship to gastrointestinal motility in LRRK2-knockout (KO) mice. The mice were subjected to acute restraint stress, and analyzed for activation of the paraventricular nucleus of the hypothalamus (PVN) using an immunohistochemical approach. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was assessed by Western blotting. The KO mice showed a lower number of c-Fos-positive cells and disruption of the ERK signaling pathway in the PVN in the presence of restraint stress. Stress responses in terms of both upper and lower gastrointestinal motility were alleviated in the mice, accompanied by lower c-Fos immunoreactivity in enteric excitatory neurons. Our present findings suggest that LRRK2 is a newly recognized molecule regulating the stress pathway in the PVN, playing a role in stress-related gastrointestinal dysmotility.

LRRK2: An Emerging New Molecule in the Enteric Neuronal System That Quantitatively Regulates Neuronal Peptides and IgA in the Gut.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) is a recently discovered molecule associated with familial and sporadic Parkinson's disease. It regulates many central neuronal functions such as cell proliferation, apoptosis, autophagy, and axonal extension. However, in contrast to the well-documented function of LRRK2 in central neurons, it is unclear whether LRRK2 is expressed in enteric neurons and affects the physiology of the gut. AIMS: By examining LRRK2-KO mice, this study investigated whether enteric neurons express LRRK2 and whether intestinal neuronal peptides and IgA are quantitatively changed. METHODS: Intestinal protein lysates and sections prepared from male C57BL/6 J mice were analyzed by Western blotting and immunostaining using anti-LRRK2 antibody, respectively. Intestinal neuronal peptide-mRNAs were quantified by real-time PCR in wild-type mice and LRRK2-KO mice. Intestinal IgA was quantified by ELISA. Lamina propria mononuclear cells (LPMCs) were analyzed by flow cytometry to evaluate the ratio of B1 to B2 B cells. RESULTS: Western analysis and immunostaining revealed that LRRK2 is expressed in enteric neurons. The amounts of mRNA for vasoactive intestinal peptide, neuropeptide Y, and substance P were increased in LRRK2-KO mice accompanied by an increment of IgA. However, the intestinal B cell subpopulations were not altered in LRRK2-KO mice. CONCLUSIONS: For the first time, we have revealed that LRRK2 is expressed in enteric neurons and related to quantitative alterations of neuronal peptide and IgA. Our study highlights the importance of LRRK2 in enteric neurons as well as central neurons.

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.

Leucine-rich repeat kinase 2 is a regulator of B cell function, affecting homeostasis, BCR signaling, IgA production, and TI antigen responses.

LRRK2 is the causal molecule of autosomal dominant familial Parkinson's disease. B2 cells express a much higher LRRK2 mRNA level than B1 cells. To reveal the function of LRRK2 in B cells, we analyzed B cell functions in LRRK2-knockout (LRRK2(-/-)) mice. LRRK2(-/-) mice had significantly higher counts of peritoneal B1 cells than wild-type mice. After BCR stimulation, phosphor-Erk1/2 of splenic B2 cells was enhanced to a higher degree in LRRK2(-/-) mice. LRRK2(-/-) mice had a significantly higher serum IgA level, and TNP-Ficoll immunization increased the titer of serum anti-TNP IgM antibody. LRRK2 may play important roles in B cells.

Elemental diet moderates 5-fluorouracil-induced gastrointestinal mucositis through mucus barrier alteration.

PURPOSE: There are reports that elemental diet (ED) ameliorates oral mucositis caused by antineoplastic chemotherapy. Although this effectiveness may be partly due to high nutrient absorption, the effects of chemotherapy on mucosal defense mechanisms remain unclear. We investigated the effects of oral supplementation with ED on mucin in 5-fluorouracil (5-FU)-induced intestinal mucositis. METHODS: 5-FU was administered to rats orally once daily, and ED was supplied orally twice daily for 5 days. The severity of mucositis was assessed by length, dry tissue weight, and villus height of the intestinal tract. Using anti-mucin monoclonal antibody, we compared the immunoreactivity in the gastrointestinal (GI) tract and mucin content by histological and biochemical examinations. RESULTS: Oral supplementation with ED reduced histological damage and loss of length, dry tissue weight, and villus height induced by 5-FU administration. ED markedly altered PGM34 antibody immunoreactivity and mucin contents in the small intestine of rats with 5-FU-induced mucositis. CONCLUSIONS: ED may possibly be more effective for the prevention of antineoplastic chemotherapy-induced mucositis through the activation of GI mucus cells.

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

Leucine-rich repeat kinase 2 (LRRK2) has been identified as the causal molecule for autosomal-dominant Parkinson's 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-3ß, and that this interaction enhances the kinase activity of GSK-3ß. Furthermore, we found that LRRK2-mediated activation of GSK-3ß 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-3ß and as a physiological regulator of neurite outgrowth and axonal transport through regulation of the GSK-3ß-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-3ß may be involved in development of pathological features in the initial stage of PD.

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

BACKGROUND: Leucine-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. RESULTS: 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. CONCLUSIONS: 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.

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.

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

LRRK2, the causal molecule of familial Parkinson's 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.

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

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinson's 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.

I(2020)T 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 Parkinson's 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.