Signal Transduction Protein Array Analysis Links LRRK2 to Ste20 Kinases and PKC Zeta That Modulate Neuronal Plasticity

substrate phosphorylation..Ste20 kinases and PKC zeta contribute to neuronal Tau phosphorylation, neurite outgrowth and synaptic plasticity under physiological conditions. Our data suggest that these kinases may also be involved in synaptic dysfunction and neurite fragmentation in transgenic mice and in human PD patients carrying toxic gain-of-function LRRK2 mutations

ARHGEF7 (BETA-PIX) Acts as Guanine Nucleotide Exchange Factor for Leucine-Rich Repeat Kinase 2

Background: Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson’s disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro. Methodology/Principal Findings: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7. Conclusions/Significance: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPas

Leucine-Rich Repeat Kinase 2 Modulates Retinoic Acid-Induced Neuronal Differentiation of Murine Embryonic Stem Cells

Background: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson’s disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment. Methodology/Principal Findings: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/2 cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2deficient stem cells in culture. Conclusion/Significance: Parkinson’s disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in othe

Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations

​Leucine-rich repeat kinase 2 (​LRRK2) mutations are the most common genetic cause of Parkinson’s disease. ​LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson’s disease, but whether ​LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that ​LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase ​αTAT1 prevents association of mutant ​LRRK2 with microtubules, and the deacetylase inhibitor ​trichostatin A (​TSA) restores axonal transport. In vivo knockdown of the deacetylases ​HDAC6 and ​Sirt2, or administration of ​TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson’s disease

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

Pilocarpine-Induced Status Epilepticus in Rats Involves Ischemic and Excitotoxic Mechanisms

The neuron loss characteristic of hippocampal sclerosis in temporal lobe epilepsy patients is thought to be the result of excitotoxic, rather than ischemic, injury. In this study, we assessed changes in vascular structure, gene expression, and the time course of neuronal degeneration in the cerebral cortex during the acute period after onset of pilocarpine-induced status epilepticus (SE). Immediately after 2 hr SE, the subgranular layers of somatosensory cortex exhibited a reduced vascular perfusion indicative of ischemia, whereas the immediately adjacent supragranular layers exhibited increased perfusion. Subgranular layers exhibited necrotic pathology, whereas the supergranular layers were characterized by a delayed (24 h after SE) degeneration apparently via programmed cell death. These results indicate that both excitotoxic and ischemic injuries occur during pilocarpine-induced SE. Both of these degenerative pathways, as well as the widespread and severe brain damage observed, should be considered when animal model-based data are compared to human pathology

The MDM2-p53 pathway is involved in preconditioning-induced neuronal tolerance to ischemia.

Brain preconditioning (PC) refers to a state of transient tolerance against a lethal insult that can be evoked by a prior mild event. It is thought that PC may induce different pathways responsible for neuroprotection, which may involve the attenuation of cell damage pathways, including the apoptotic cell death. In this context, p53 is a stress sensor that accumulates during brain ischemia leading to neuronal death. The murine double minute 2 gene (MDM2), a p53-specific E3 ubiquitin ligase, is the main cellular antagonist of p53, mediating its degradation by the proteasome. Here, we study the role of MDM2-p53 pathway on PC-induced neuroprotection both in cultured neurons (in vitro) and rat brain (in vivo). Our results show that PC increased neuronal MDM2 protein levels, which prevented ischemiainduced p53 stabilization and neuronal death. Indeed, PC attenuated ischemia-induced activation of the p53/PUMA/caspase-3 signaling pathway. Pharmacological inhibition of MDM2-p53 interaction in neurons abrogated PC-induced neuroprotection against ischemia. Finally, the relevance of the MDM2-p53 pathway was confirmed in rat brain using a PC model in vivo. These findings demonstrate the key role of the MDM2-p53 pathway in PC-induced neuroprotection against a subsequent ischemic insult and poses MDM2 as an essential target in ischemic tolerance

Non-Motor and Motor Features in LRRK2 Transgenic Mice

10.1371/journal.pone.0070249PLoS ONE87-POLN

Comparative mitochondrial proteomics: perspective in human diseases

Mitochondria are the most complex and the most important organelles of eukaryotic cells, which are involved in many cellular processes, including energy metabolism, apoptosis, and aging. And mitochondria have been identified as the "hot spot" by researchers for exploring relevant associated dysfunctions in many fields. The emergence of comparative proteomics enables us to have a close look at the mitochondrial proteome in a comprehensive and effective manner under various conditions and cellular circumstances. Two-dimensional electrophoresis combined with mass spectrometry is still the most popular techniques to study comparative mitochondrial proteomics. Furthermore, many new techniques, such as ICAT, MudPIT, and SILAC, equip researchers with more flexibilities inselecting proper methods. This article also reviews the recent development of comparative mitochondrial proteomics on diverse human diseases. And the results of mitochondrial proteomics enhance a better understanding of the pathogenesis associated with mitochondria and provide promising therapeutic targets

Transcriptome Analysis of the Hippocampal CA1 Pyramidal Cell Region after Kainic Acid-Induced Status Epilepticus in Juvenile Rats

Molecular mechanisms involved in epileptogenesis in the developing brain remain poorly understood. The gene array approach could reveal some of the factors involved by allowing the identification of a broad scale of genes altered by seizures. In this study we used microarray analysis to reveal the gene expression profile of the laser microdissected hippocampal CA1 subregion one week after kainic acid (KA)-induced status epilepticus (SE) in 21-day-old rats, which are developmentally roughly comparable to juvenile children. The gene expression analysis with the Chipster software generated a total of 1592 differently expressed genes in the CA1 subregion of KA-treated rats compared to control rats. The KEGG database revealed that the identified genes were involved in pathways such as oxidative phosporylation (26 genes changed), and long-term potentiation (LTP; 18 genes changed). Also genes involved in Ca2+ homeostasis, gliosis, inflammation, and GABAergic transmission were altered. To validate the microarray results we further examined the protein expression for a subset of selected genes, glial fibrillary protein (GFAP), apolipoprotein E (apo E), cannabinoid type 1 receptor (CB1), Purkinje cell protein 4 (PEP-19), and interleukin 8 receptor (CXCR1), with immunohistochemistry, which confirmed the transcriptome results. Our results showed that SE resulted in no obvious CA1 neuronal loss, and alterations in the expression pattern of several genes during the early epileptogenic phase were comparable to previous gene expression studies of the adult hippocampus of both experimental epileptic animals and patients with temporal lobe epilepsy (TLE). However, some changes seem to occur after SE specifically in the juvenile rat hippocampus. Insight of the SE-induced alterations in gene expression and their related pathways could give us hints for the development of new target-specific antiepileptic drugs that interfere with the progression of the disease in the juvenile age group