Mutant-TMEM230-induced neurodegeneration and impaired axonal mitochondrial transport.

Parkinson's disease (PD) is a neurodegenerative disease with movement disorders including resting tremor, rigidity, bradykinesia and postural instability. Recent studies have identified a new PD associated gene, TMEM230 (transmembrane protein 230). However, the pathological roles of TMEM230 and its variants are not fully understood. TMEM230 gene encodes two protein isoforms. Isoform2 is the major protein form (~95%) in human. In this study, we overexpress isoform2 TMEM230 variants (WT or PD-linked *184Wext*5 mutant) or knockdown endogenous protein in cultured SH-5Y5Y cells and mouse primary hippocampus neurons to study their pathological roles. We found that overexpression of WT and mutant TMEM230 or knockdown of endogenous TMEM230-induced neurodegeneration and impaired mitochondria transport at the retrograde direction in axons. Mutant TMEM230 caused more severe neurotoxicity and mitochondrial transport impairment than WT-TMEM230 did. Our results demonstrate that maintaining TMEM230 protein levels is critical for neuron survival and axon transport. These findings suggest that mutant-TMEM230-induced mitochondrial transport impairment could be the early event leading to neurite injury and neurodegeneration in PD development.

AMPK signaling mediates synphilin-1-induced hyperphagia and obesity in Drosophila.

Expression of synphilin-1 in neurons induces hyperphagia and obesity in a Drosophila model. However, the molecular pathways underlying synphilin-1-linked obesity remain unclear. Here, Drosophila models and genetic tools were used to study the synphilin-1-linked pathways in energy balance by combining molecular biology and pharmacological approaches. We found that expression of human synphilin-1 in flies increased AMP-activated kinase (AMPK) phosphorylation at Thr172 compared with that in non-transgenic flies. Knockdown of AMPK reduced AMPK phosphorylation and food intake in non-transgenic flies, and further suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia, fat storage and body weight gain in transgenic flies. Expression of constitutively activated AMPK significantly increased food intake and body weight gain in non-transgenic flies, but it did not alter food intake in the synphilin-1 transgenic flies. In contrast, expression of dominant-negative AMPK reduced food intake in both non-transgenic and synphilin-1 transgenic flies. Treatment with STO-609 also suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia and body weight gain. These results demonstrate that the AMPK signaling pathway plays a critical role in synphilin-1-induced hyperphagia and obesity. These findings provide new insights into the mechanisms of synphilin-1-controlled energy homeostasis.

G2019S-LRRK2 mutation enhances MPTP-linked Parkinsonism in mice.

Parkinson's disease (PD) is a common neurodegenerative disease with a heterogeneous etiology that involves genetic and environmental factors or exogenous. Current LRRK2 PD animal models only partly reproduce the characteristics of the disease with very subtle dopaminergic neuron degeneration. We developed a new model of PD that combines a sub-toxic MPTP insult to the G2019S-LRRK2 mutation. Our newly generated mice, overexpressing mutant G2019S-LRRK2 protein in the brain, displayed a mild, age-dependent progressive motor impairment, but no reduction of lifespan. Cortical neurons from G2019S-LRRK2 mice showed an increased vulnerability to stress insults, compared with neurons overexpressing wild-type WT-LRRK2, or non-transgenic (nTg) neurons. The exposure of LRRK2 transgenic mice to a sub-toxic dose of MPTP resulted in severe motor impairment, selective loss of dopamine neurons and increased astrocyte activation, whereas nTg mice with MPTP exposure showed no deficits. Interestingly, mice overexpressing WT-LRRK2 showed a significant impairment that was milder than for the mutant G2019S-LRRK2 mice. L-DOPA treatments could partially improve the movement impairments but did not protect the dopamine neuron loss. In contrast, treatments with an LRRK2 kinase inhibitor significantly reduced the dopaminergic neuron degeneration in this interaction model. Our studies provide a novel LRRK2 gene-MPTP interaction PD mouse model, and a useful tool for future studies of PD pathogenesis and therapeutic intervention.

GTP-binding inhibitors increase LRRK2-linked ubiquitination and Lewy body-like inclusions.

Parkinson's disease (PD) is one of the most common movement disorders with loss of dopaminergic neurons and the presence of Lewy bodies in certain brain areas. However, it is not clear how Lewy body (inclusion with protein aggregation) formation occurs. Mutations in leucine-rich repeat kinase 2 (LRRK2) can cause a genetic form of PD and contribute to sporadic PD with the typical Lewy body pathology. Here, we used our recently identified LRRK2 GTP-binding inhibitors as pharmacological probes to study the LRRK2-linked ubiquitination and protein aggregation. Pharmacological inhibition of GTP-binding by GTP-binding inhibitors (68 and Fx2149) increased LRRK2-linked ubiquitination predominantly via K27 linkage. Compound 68- or Fx2149 increased G2019S-LRRK2-linked ubiquitinated aggregates, which occurred through the atypical linkage types K27 and K63. Coexpression of K27R and K63R, which prevented ubiquitination via K27 and K63 linkages, reversed the effects of 68 and Fx2149. Moreover, 68 and Fx2149 also promoted G2019S-LRRK2-linked aggresome (Lewy body-like inclusion) formation via K27 and K63 linkages. These findings demonstrate that LRRK2 GTP-binding activity is critical in LRRK2-linked ubiquitination and aggregation formation. These studies provide novel insight into the LRRK2-linked Lewy body-like inclusion formation underlying PD pathogenesis.

Synphilin-1 Interacts with AMPK and Increases AMPK Phosphorylation.

A role for the cytoplasmic protein synphilin-1 in regulating energy balance has been demonstrated recently. Expression of synphilin-1 increases ATP levels in cultured cells. However, the mechanism by which synphilin-1 alters cellular energy status is unknown. Here, we used cell models and biochemical approaches to investigate the cellular functions of synphilin-1 on the AMP-activated protein kinase (AMPK) signaling pathway, which may affect energy balance. Overexpression of synphilin-1 increased AMPK phosphorylation (activation). Moreover, synphilin-1 interacted with AMPK by co-immunoprecipitation and GST (glutathione S-transferase) pull-down assays. Knockdown of synphilin-1 reduced AMPK phosphorylation. Overexpression of synphilin-1 also altered AMPK downstream signaling, i.e., a decrease in acetyl CoA carboxylase (ACC) phosphorylation, and an increase in p70S6K phosphorylation. Treatment of compound C (an AMPK inhibitor) reduced synphilin-1 binding with AMPK. In addition, compound C diminished synphilin-1-induced AMPK phosphorylation, and the increase in cellular ATP (adenosine triphosphate) levels. Our results demonstrated that synphilin-1 couples with AMPK, and they exert mutual effects on each other to regulate cellular energy status. These findings not only identify novel cellular actions of synphilin-1, but also provide new insights into the roles of synphilin-1 in regulating energy currency, ATP.

Synphilin-1 attenuates mutant LRRK2-induced neurodegeneration in Parkinson's disease models.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinsonism with pleomorphic pathology including deposits of aggregated protein and neuronal degeneration. The pathogenesis of LRRK2-linked Parkinson's disease (PD) is not fully understood. Here, using co-immunoprecipitation, we found that LRRK2 interacted with synphilin-1 (SP1), a cytoplasmic protein that interacts with α-synuclein and has implications in PD pathogenesis. LRRK2 interacted with the N-terminus of SP1 whereas SP1 predominantly interacted with the C-terminus of LRRK2, including kinase domain. Co-expression of SP1 with LRRK2 increased LRRK2-induced cytoplasmic aggregation in cultured cells. Moreover, SP1 also attenuated mutant LRRK2-induced toxicity and reduced LRRK2 kinase activity in cultured cells. Knockdown of SP1 by siRNA enhanced LRRK2 neuronal toxicity. In vivo Drosophila studies, co-expression of SP1 and mutant G2019S-LRRK2 in double transgenic Drosophila increased survival and improved locomotor activity. Expression of SP1 protects against G2019S-LRRK2-induced dopamine neuron loss and reduced LRRK2 phosphorylation in double transgenic fly brains. Our findings demonstrate that SP1 attenuates mutant LRRK2-induced PD-like phenotypes and plays a neural protective role.

The Drosophila hep pathway mediates Lrrk2-induced neurodegeneration.

Although the pathogenesis of Parkinson's disease (PD) remains unclear, mutations in leucine-rich repeat kinase 2 (Lrrk2) are among the major causes of familial PD. Most of these mutations disrupt Lrrk2 kinase and (or) GTPase domain function, resulting in neuronal degeneration. However, the signal pathways underlying Lrrk2-induced neuronal degeneration are not fully understood. There is an expanding body of evidence that suggests a link between Lrrk2 function and MAP kinase (MAPK) cascades. To further investigate this link in vivo, genetic RNAi screens of the MAPK pathways were performed in a Drosophila model to identify genetic modifier(s) that can suppress G2019S-Lrrk2-induced PD-like phenotypes. The results revealed that the knockdown of hemipterous (hep, or JNKK) increased fly survival time, improved locomotor function, and reduced loss of dopaminergic neurons in G2019S-Lrrk2 transgenic flies. Expression of the dominant-negative allele of JNK (JNK-DN), a kinase that is downstream of hep in G2019S-Lrrk2 transgenic flies, elicited a similar effect. Moreover, treatment with the JNK inhibitor SP600125 partially reversed the G2019S-Lrrk2-induced loss of dopaminergic neurons. These results indicate that the hep pathway plays an important role in Lrrk2-linked Parkinsonism in flies. These studies provide new insights into the molecular mechanisms underlying Lrrk2-linked PD pathogenesis and aid in identifying potential therapeutic targets.

Novel LRRK2 GTP-binding inhibitors reduced degeneration in Parkinson's disease cell and mouse models.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. LRRK2 contains Guanosine-5'-triphosphate (GTP) binding, GTPase and kinase activities that have been implicated in the neuronal degeneration of PD pathogenesis, making LRRK2, a potential drug target. To date, there is no disease-modifying drug to slow the neuronal degeneration of PD and no published LRRK2 GTP domain inhibitor. Here, the biological functions of two novel GTP-binding inhibitors of LRRK2 were examined in PD cell and mouse models. Through a combination of computer-aided drug design (CADD) and LRRK2 bio-functional screens, two novel compounds, 68: and 70: , were shown to reduce LRRK2 GTP binding and to inhibit LRRK2 kinase activity in vitro and in cultured cell assays. Moreover, these two compounds attenuated neuronal degeneration in human SH-SY5Y neuroblastoma cells and mouse primary neurons expressing mutant LRRK2 variants. Although both compounds inhibited LRRK2 kinase activity and reduced neuronal degeneration, solubility problems with 70: prevented further testing in mice. Thus, only 68: was tested in a LRRK2-based lipopolysaccharide (LPS)-induced pre-inflammatory mouse model. 68: reduced LRRK2 GTP-binding activity and kinase activity in brains of LRRK2 transgenic mice after intraperitoneal injection. Moreover, LPS induced LRRK2 upregulation and microglia activation in mouse brains. These findings suggest that disruption of GTP binding to LRRK2 represents a potential novel therapeutic approach for PD intervention and that these novel GTP-binding inhibitors provide both tools and lead compounds for future drug development.

MSC-Derived Extracellular Vesicles Alleviate NLRP3/GSDMD-Mediated Neuroinflammation in Mouse Model of Sporadic Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease, with sporadic form being the predominant type. Neuroinflammation plays a critical role in accelerating pathogenic processes in AD. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEVs) regulate inflammatory responses and show great promise for treating AD. Induced pluripotent stem cell (iPSC)-derived MSCs are similar to MSCs and exhibit low immunogenicity and heterogeneity, making them promising cell sources for clinical applications. This study examined the anti-inflammatory effects of MSC-sEVs in a streptozotocin-induced sporadic mouse model of AD (sAD). The intracisternal administration of iPSC-MSC-sEVs alleviated NLRP3/GSDMD-mediated neuroinflammation, decreased amyloid deposition and neuronal apoptosis, and mitigated cognitive dysfunction. Furthermore, it explored the role of miR-223-3p in the iPSC-MSC-sEVs-mediated anti-inflammatory effects in vitro. miR-223-3p directly targeted NLRP3, whereas inhibiting miR-223-3p almost completely reversed the suppression of NLRP3 by MSC-sEVs, suggesting that miR-223-3p may, at least partially, account for MSC-sEVs-mediated anti-inflammation. Results obtained suggest that intracisternal administration of iPSC-MSC-sEVs can reduce cognitive impairment by inhibiting NLRP3/GSDMD neuroinflammation in a sAD mouse model. Therefore, the present study provides a proof-of-principle for applying iPSC-MSC-sEVs to target neuroinflammation in sAD.

Inhibitors of LRRK2 kinase attenuate neurodegeneration and Parkinson-like phenotypes in Caenorhabditis elegans and Drosophila Parkinson's disease models.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinson's disease (PD) and have also been found in the more common sporadic form of PD, thus positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD.

LRRK2 kinase activity mediates toxic interactions between genetic mutation and oxidative stress in a Drosophila model: suppression by curcumin.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. The pathogenesis of PD is believed to involve both genetic susceptibility and environmental factors. Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause genetic forms of PD, and the LRRK2 locus contributes to sporadic PD. Environmental toxins are believed to act in part by causing oxidative stress. Here we employed cell and Drosophila models to investigate the interaction between LRRK2 genetic mutations and oxidative stress. We found that H(2)O(2) increased LRRK2 kinase activity and enhanced LRRK2 cell toxicity in cultured cells and mouse primary cortical neurons. Furthermore, a sub-toxic dose of H(2)O(2) significantly shortened the survival of LRRK2 transgenic flies and augmented LRRK2-induced locomotor defects and dopamine neuron loss. Treatment with a LRRK2 kinase inhibitor (GW5074) or an anti-oxidant (curcumin) significantly suppressed these PD-like phenotypes in flies. Moreover, curcumin significantly reduced LRRK2 kinase activity and the levels of oxidized proteins, and thus acted as not only an antioxidant but also a LRRK2 kinase inhibitor. These results indicate that LRRK2 genetic alterations can interact with oxidative stress, converging on a pathogenic pathway that may be related to PD. These studies also identified curcumin as a LRRK2 kinase inhibitor that may be a useful candidate for LRRK2-linked PD intervention.

Synphilin-1 attenuates neuronal degeneration in the A53T alpha-synuclein transgenic mouse model.

Genetic alterations in alpha-synuclein cause autosomal dominant familial Parkinsonism and may contribute to sporadic Parkinson's disease (PD). Synphilin-1 is an alpha-synuclein-interacting protein, with implications in PD pathogenesis related to protein aggregation. Currently, the in vivo role of synphilin-1 in alpha-synuclein-linked pathogenesis is not fully understood. Using the mouse prion protein promoter, we generated synphilin-1 transgenic mice, which did not display PD-like phenotypes. However, synphilin-1/A53T alpha-synuclein double-transgenic mice survived longer than A53T alpha-synuclein single-transgenic mice. There were attenuated A53T alpha-synuclein-induced motor abnormalities and decreased astroglial reaction and neuronal degeneration in brains in double-transgenic mice. Overexpression of synphilin-1 decreased caspase-3 activation, increased beclin-1 and LC3 II expression and promoted formation of aggresome-like structures, suggesting that synphilin-1 alters multiple cellular pathways to protect against neuronal degeneration. These studies demonstrate that synphilin-1 can diminish the severity of alpha-synucleinopathy and play a neuroprotective role against A53T alpha-synuclein toxicity in vivo.

CHIP regulates leucine-rich repeat kinase-2 ubiquitination, degradation, and toxicity.

Mutation in leucine-rich repeat kinase-2 (LRRK2) is the most common cause of late-onset Parkinson's disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD.

Curcumin protects against A53T alpha-synuclein-induced toxicity in a PC12 inducible cell model for Parkinsonism.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. The pathogenesis of PD remains incompletely understood, but it appears to involve both genetic susceptibility and environmental factors. Treatment for PD that prevents neuronal death in the dopaminergic system and abnormal protein deposition in the brain is not yet available. Evidence from human and animal studies has suggested that oxidative damage critically contributes to neuronal loss in PD. Here we test whether curcumin, a potent antioxidant compound, derived from the curry spice turmeric, can protect against mutant A53T α-synuclein-induced cell death. We used PC12 cells that inducibly express A53T α-synuclein. We found that curcumin protected against A53T α-synuclein-induced cell death in a dose-dependent manner. We further found that curcumin can reduce mutant α- synuclein-induced intracellular reactive oxygen species (ROS) levels, mitochondrial depolarization, cytochrome c release, and caspase-9 and caspase-3 activation. This study demonstrate that curcumin protected against A53T mutant α-synuclein-induced cell death via inhibition of oxidative stress and the mitochondrial cell death pathway, suggesting that curcumin may be a candidate neuroprotective agent for A53T α-synuclein-linked Parkinsonism, and possibly for other genetic or sporadic forms of PD.

A Drosophila model for LRRK2-linked parkinsonism.

Mutations in the leucine-rich repeat kinase (LRRK2) gene cause late-onset autosomal dominant Parkinson's disease (PD) with pleiomorphic pathology. Previously, we and others found that expression of mutant LRRK2 causes neuronal degeneration in cell culture. Here we used the GAL4/UAS system to generate transgenic Drosophila expressing either wild-type human LRRK2 or LRRK2-G2019S, the most common mutation associated with PD. Expression of either wild-type human LRRK2 or LRRK2-G2019S in the photoreceptor cells caused retinal degeneration. Expression of LRRK2 or LRRK2-G2019S in neurons produced adult-onset selective loss of dopaminergic neurons, locomotor dysfunction, and early mortality. Expression of mutant G2019S-LRRK2 caused a more severe parkinsonism-like phenotype than expression of equivalent levels of wild-type LRRK2. Treatment with l-DOPA improved mutant LRRK2-induced locomotor impairment but did not prevent the loss of tyrosine hydroxylase-positive neurons. To our knowledge, this is the first in vivo"gain-of-function" model which recapitulates several key features of LRRK2-linked human parkinsonism. These flies may provide a useful model for studying LRRK2-linked pathogenesis and for future therapeutic screens for PD intervention.

Activation of Nrf2 in Astrocytes Suppressed PD-Like Phenotypes via Antioxidant and Autophagy Pathways in Rat and Drosophila Models.

The oxidative-stress-induced impairment of autophagy plays a critical role in the pathogenesis of Parkinson's disease (PD). In this study, we investigated whether the alteration of Nrf2 in astrocytes protected against 6-OHDA (6-hydroxydopamine)- and rotenone-induced PD-like phenotypes, using 6-OHDA-induced rat PD and rotenone-induced Drosophila PD models. In the PD rat model, we found that Nrf2 expression was significantly higher in astrocytes than in neurons. CDDO-Me (CDDO methyl ester, an Nrf2 inducer) administration attenuated PD-like neurodegeneration mainly through Nrf2 activation in astrocytes by activating the antioxidant signaling pathway and enhancing autophagy in the substantia nigra and striatum. In the PD Drosophila model, the overexpression of Nrf2 in glial cells displayed more protective effects than such overexpression in neurons. Increased Nrf2 expression in glial cells significantly reduced oxidative stress and enhanced autophagy in the brain tissue. The administration of the Nrf2 inhibitor ML385 reduced the neuroprotective effect of Nrf2 through the inhibition of the antioxidant signaling pathway and autophagy pathway. The autophagy inhibitor 3-MA partially reduced the neuroprotective effect of Nrf2 through the inhibition of the autophagy pathway, but not the antioxidant signaling pathway. Moreover, Nrf2 knockdown caused neurodegeneration in flies. Treatment with CDDO-Me attenuated the Nrf2-knockdown-induced degeneration in the flies through the activation of the antioxidant signaling pathway and increased autophagy. An autophagy inducer, rapamycin, partially rescued the neurodegeneration in Nrf2-knockdown Drosophila by enhancing autophagy. Our results indicate that the activation of the Nrf2-linked signaling pathways in glial cells plays an important neuroprotective role in PD models. Our findings not only provide a novel insight into the mechanisms of Nrf2-antioxidant-autophagy signaling, but also provide potential targets for PD interventions.

Controversy of TMEM230 Associated with Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disease with movement disorders including resting tremor, bradykinesia, rigidity, and postural instability. The key pathological features of PD are selective loss of dopaminergic (DA) neurons in substantial nigra and the presence of Lewy bodies (LBs). Mutations in TMEM230 (transmembrane protein 230) have been recently reported to play a pathological role and contribute to PD pathogenesis. TMEM230 gene encodes two isoforms of TMEM230 proteins, isoform I (183 amino acids) and isoform II (120 amino acids). The function of TMEM230 is not clear, but it may be involved in vesicle trafficking and recycling, autophagy, protein aggregation, and cell toxicity. There are four reported PD-linked TMEM230 mutations (p.Y92C, p.R141L, p.*184Wext*5, p.*184PGext*5). TMEM230-linked PD cases exhibit late-onset, good-response to levodopa, and typical clinical features of sporadic PD with DA neuronal loss in substantial nigra and Lewy body pathology. In this mini review, we recap the current literature of TMEM230 in genetic, neurobiological, and pathological studies in order to further understand the potential roles of TMEM230 in PD pathogenesis.

Curcumin Reduced H2O2- and G2385R-LRRK2-Induced Neurodegeneration.

Mutations in leucine-rich repeat kinase 2 gene (LRRK2) are the most frequent genetic factors contributing to Parkinson's disease (PD). G2385R-LRRK2 increases the risk for PD susceptibility in the Chinese population. However, the pathological role of G2385R-LRRK2 is not clear. In this study, we investigate the roles of G2385R-LRRK2 in neurodegeneration underlying PD pathogenesis using cell biology and pharmacology approaches. We demonstrated that expression of G2385R-LRRK2-induced neurotoxicity in human neuroblastoma SH-SY5Y and mouse primary neurons. G2385R-LRRK2 increased mitochondrial ROS, activates caspase-3/7, and increased PARP cleavage, resulting in neurotoxicity. Treatment with curcumin (an antioxidant) significantly protected against G2385R-LRRK2-induced neurodegeneration by reducing mitochondrial ROS, caspase-3/7 activation, and PARP cleavage. We also found that the cellular environmental stressor, H2O2 significantly promotes both WT-LRRK2- and G2385R-LRRK2-induced neurotoxicity by increasing mitochondrial ROS, caspase-3/7 activation, and PARP cleavage, while curcumin attenuated this combined neurotoxicity. These findings not only provide a novel understanding of G2385R roles in neurodegeneration and environment interaction but also provide a pharmacological approach for intervention for G2385R-LRRK2-linked PD.

A LRRK2 GTP Binding Inhibitor, 68, Reduces LPS-Induced Signaling Events and TNF-α Release in Human Lymphoblasts.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. Common genetic variation in LRRK2 modifies susceptibility to immunological disorders including Crohn's disease and leprosy. Previous studies have reported that LRRK2 is expressed in B lymphocytes and macrophages, suggesting a role for LRRK2 in immunological functions. In this study, we characterized the LRRK2 protein expression and phosphorylation using human lymphoblasts. Lipopolysaccharide (LPS), a proinflammatory agent, induced the increase of LRRK2 expression and kinase activities in human lymphoblasts in a time-dependent manner. Moreover, LPS activated the Toll-like receptor (TLR) signaling pathway, increased TRAF6/LRRK2 interaction, and elevated the phosphorylation levels of MAPK (JNK1/2, p38, and ERK1/2) and IkBα. Treatment with LRRK2 inhibitor 68 reduced LPS-induced TRAF6/LRRK2 interaction and MAPK and IkBα phosphorylation, thereby reducing TNF-α secretion. These results indicate that LRRK2 is actively involved in proinflammatory responses in human lymphoblasts, and inhibition of GTP binding by 68 results in an anti-inflammation effect against proinflammatory stimuli. These findings not only provide novel insights into the mechanisms of LRRK2-linked immune and inflammatory responses in B-cell-like lymphoblasts, but also suggest that 68 may also have potential therapeutic value for LRRK2-linked immunological disorders.

Baicalein reduces E46K alpha-synuclein aggregation in vitro and protects cells against E46K alpha-synuclein toxicity in cell models of familiar Parkinsonism.

The E46K is a point mutation in alpha-synuclein (alpha-syn) that causes familial Parkinsonism with Lewy body dementia. We have now generated a cell model of Parkinsonism/Parkinson's disease (PD) and demonstrated cell toxicity after expression of E46K in the differentiated PC12 cells. E46K alpha-syn inhibited proteasome activity and induced mitochondrial depolarization in the cell model. Baicalein has been reported to inhibit fibrillation of wild type alpha-syn in vitro, and to protect neurons against several chemical-induced models of PD. We now report that baicalein significantly attenuated E46K-induced mitochondrial depolarization and proteasome inhibition, and protected cells against E46K-induced toxicity in a cell model of PD. Baicalein also reduced E46K fibrilization in vitro, with a concentration-dependent decrease in beta sheet conformation, though it increased some oligomeric species, and decreased formation of E46K alpha-syn-induced aggregates and rescued toxicity in N2A cells. Taken together, these data indicate that mitochondrial dysfunction, proteasome inhibition and specific aspects of abnormal E46K aggregation accompany E46K alpha-syn-induced cell toxicity, and baicalein can protect as well as altering aggregation properties. Baicalein has potential as a tool to understand the relation between different aggregation species and toxicity, and might be a candidate compound for further validation by using in vivo alpha-syn genetic PD models.