Mutually exclusive antiproliferative effect of cell line-specific HOX inhibition in epithelial ovarian cancer cell lines: SKOV-3 vs RMUG-S.

We aimed to discover cell line-specific overexpressed HOX genes responsible for chemoresistance and to identify the mechanisms behind HOX-induced cell line-specific chemoresistance in EOC. Ten HOX genes and eight EOC cell lines were tested for any cell line-specific overexpression that presents a mutually exclusive pattern. Cell viability was evaluated after treatment with cisplatin and/or siRNA for cell line-specific overexpressed HOX genes. Immunohistochemical (IHC) staining for HOXB9 was performed in 84 human EOC tissues. HOXA10 and HOXB9 were identified as cell line-specific overexpressed HOX genes for SKOV-3 and RMUG-S, respectively. Inhibiting the expression of cell line-specific HOX genes, but not of other HOX genes, significantly decreased cell viability. In SKOV-3 cells, cell viability decreased to 46.5% after initial 10 µM cisplatin treatment; however, there was no further decrease upon additional treatment with HOXA10 siRNA. In contrast, cell viability did not significantly decrease upon cisplatin treatment in RMUG-S cells, but decreased to 65.5% after additional treatment with HOXB9 siRNA. In both cell lines, inhibiting cell line-specific HOX expression enhanced apoptosis but suppressed the expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, MMP9, and Oct4. IHC analysis showed that platinum-resistant cancer tissues more frequently had high HOXB9 expression than platinum-sensitive cancer tissues. HOXB9, which is overexpressed in RMUG-S but not in SKOV-3 cells, appeared to be associated with cell line-specific platinum resistance in RMUG-S. Inhibiting HOXB9 overexpression in RMUG-S cells may effectively eliminate platinum-resistant ovarian cancer cells by facilitating apoptosis and inhibiting EMT.

Cereblon Maintains Synaptic and Cognitive Function by Regulating BK Channel.

Mutations in the cereblon (CRBN) gene cause human intellectual disability, one of the most common cognitive disorders. However, the molecular mechanisms of CRBN-related intellectual disability remain poorly understood. We investigated the role of CRBN in synaptic function and animal behavior using male mouse and Drosophila models. Crbn knock-out (KO) mice showed normal brain and spine morphology as well as intact synaptic plasticity; however, they also exhibited decreases in synaptic transmission and presynaptic release probability exclusively in excitatory synapses. Presynaptic function was impaired not only by loss of CRBN expression, but also by expression of pathogenic CRBN mutants (human R419X mutant and Drosophila G552X mutant). We found that the BK channel blockers paxilline and iberiotoxin reversed this decrease in presynaptic release probability in Crbn KO mice. In addition, paxilline treatment also restored normal cognitive behavior in Crbn KO mice. These results strongly suggest that increased BK channel activity is the pathological mechanism of intellectual disability in CRBN mutations.SIGNIFICANCE STATEMENTCereblon (CRBN), a well known target of the immunomodulatory drug thalidomide, was originally identified as a gene that causes human intellectual disability when mutated. However, the molecular mechanisms of CRBN-related intellectual disability remain poorly understood. Based on the idea that synaptic abnormalities are the most common factor in cognitive dysfunction, we monitored the synaptic structure and function of Crbn knock-out (KO) animals to identify the molecular mechanisms of intellectual disability. Here, we found that Crbn KO animals showed cognitive deficits caused by enhanced BK channel activity and reduced presynaptic glutamate release. Our findings suggest a physiological pathomechanism of the intellectual disability-related gene CRBN and will contribute to the development of therapeutic strategies for CRBN-related intellectual disability.

Distinct synaptic vesicle recycling in inhibitory nerve terminals is coordinated by SV2A.

Proper brain function requires a balance between excitatory and inhibitory neuronal activity. This balance, which is disrupted in various neural disorders, ultimately depends on the functional properties of both excitatory and inhibitory neurons; however, how the physiological properties of presynaptic terminals are controlled in these neurons is largely unknown. In this study, we generated pHluorin-conjugated, synaptic vesicle-specific tracers that are preferentially expressed in excitatory or inhibitory nerve terminals. We found that synaptic vesicle recycling is ∼1.8-fold slower in inhibitory nerve terminals than excitatory nerve terminals, resulting in reduced efficacy of synaptic transmission in inhibitory presynaptic terminals during repetitive activities. Interestingly, this relative difference in trafficking efficiency is mediated by synaptic vesicle protein 2A (SV2A), which is more highly expressed in inhibitory synapses and differentially controls sorting of synaptic protein, synaptotagmin I. These findings indicate that SV2A coordinates distinct properties of synaptic vesicle recycling between excitatory and inhibitory synapses.

Role of limonin in anticancer effects of Evodia rutaecarpa on ovarian cancer cells.

BACKGROUND: Ovarian cancer therapy generally involves systemic chemotherapy with anticancer drugs; however, chemotherapy with a platinum-based drug has often been shown to cause adverse reactions and drug resistance in ovarian cancer patients. Evodia rutaecarpa (ER) reportedly shows anticancer activity against various types of cancer cells. However, the effects of ER have not yet been fully uncovered in ovarian cancer. METHODS: In the present study, we investigated the anticancer effects of an ER extract and its components against the ovarian cancer cell lines SKOV-33, A2780, RMUG-S and a cisplatin-resistant SKOV-3 cell line (CisR SKOV-3). Cell viability and colony formation assays along with subcellular fractionation analysis, immunoblotting, and immunofluorescence staining were performed. RESULTS: ER treatment led to a significant reduction in the viability of SKOV-3 cells. Moreover, limonin, a compound found in ER, reduced the viability of both serous-type (SKOV-3 and A2780) and mucinous-type (RMUG-S) ovarian cancer cells by inducing apoptosis via activation of the p53 signaling pathway. Furthermore, limonin reversed the drug resistance through activation of apoptosis in CisR SKOV-3. CONCLUSION: Taken together, our findings suggest that limonin contributes to the anti-ovarian cancer effects of ER by inducing apoptosis via activation of the p53 signaling pathway.

Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration.

BACKGROUND: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo. METHODS: In vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35. RESULTS: Our screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo. CONCLUSIONS: Here we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

LRRK2 kinase regulates α-synuclein propagation via RAB35 phosphorylation.

Propagation of α-synuclein aggregates has been suggested as a contributing factor in Parkinson's disease (PD) progression. However, the molecular mechanisms underlying α-synuclein aggregation are not fully understood. Here, we demonstrate in cell culture, nematode, and rodent models of PD that leucine-rich repeat kinase 2 (LRRK2), a PD-linked kinase, modulates α-synuclein propagation in a kinase activity-dependent manner. The PD-linked G2019S mutation in LRRK2, which increases kinase activity, enhances propagation efficiency. Furthermore, we show that the role of LRRK2 in α-synuclein propagation is mediated by RAB35 phosphorylation. Constitutive activation of RAB35 overrides the reduced α-synuclein propagation phenotype in lrk-1 mutant C. elegans. Finally, in a mouse model of synucleinopathy, administration of an LRRK2 kinase inhibitor reduced α-synuclein aggregation via enhanced interaction of α-synuclein with the lysosomal degradation pathway. These results suggest that LRRK2-mediated RAB35 phosphorylation is a potential therapeutic target for modifying disease progression.

Synapses in neurodegenerative diseases.

Synapse is the basic structural and functional component for neural communication in the brain. The presynaptic terminal is the structural and functionally essential area that initiates communication and maintains the continuous functional neural information flow. It contains synaptic vesicles (SV) filled with neurotransmitters, an active zone for release, and numerous proteins for SV fusion and retrieval. The structural and functional synaptic plasticity is a representative characteristic; however, it is highly vulnerable to various pathological conditions. In fact, synaptic alteration is thought to be central to neural disease processes. In particular, the alteration of the structural and functional phenotype of the presynaptic terminal is a highly significant evidence for neural diseases. In this review, we specifically describe structural and functional alteration of nerve terminals in several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). [BMB Reports 2017; 50(5): 237-246].

Impairment of SOD1-G93A motility is linked to mitochondrial movement in axons of hippocampal neurons.

Superoxide dismutase 1 (SOD1) is a well-known antioxidant enzyme. Mutation of SOD1 is closely associated with the pathogenesis of neurodegenerative disorders, such as amyotrophic lateral sclerosis and Alzheimer's disease. However, the pathologic pathways linking neurodegenerative diseases with mutation of SOD1 remain elusive. Here, we investigated the motility of SOD1-WT and -G93A (a pathogenic mutant of SOD1), and observed correlation of axonal transport of the mutant protein with mitochondria in primary cultured hippocampal neurons. The SOD1-G93A mutant showed significant accumulation at vGlut1-positive synaptic boutons and in cell bodies, compared to SOD1-WT. The proportions of motile WT and G93A proteins were similar (~30 %) while the motility velocity of SOD1-G93A was significantly slower (~40 %) than that of the WT counterpart. This motility defect of SOD1-G93A was highly correlated with mitochondrial movement. Our results collectively suggest that the SOD1-G93A mutant has a defect in motility that is linked to mitochondrial transport in axons.

Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses.

Synaptic vesicle retrieval is an essential process for continuous maintenance of neural information flow after synaptic transmission. Epsin1, originally identified as an EPS15-interacting protein, is a major component of clathrin-mediated endocytosis. However, the role of Epsin1 in synaptic vesicle endocytosis at CNS synapses remains elusive. Here, we showed significantly altered synaptic vesicle endocytosis in neurons transfected with shRNA targeting Epsin1 during/after neural activity. Endocytosis was effectively restored by introducing shRNA-insensitive Epsin1 into Epsin1-depleted neurons. Domain studies performed on neurons in which domain deletion mutants of Epsin1 were introduced after Epsin1 knockdown revealed that ENTH, CLAP, and NPFs are essential for synaptic vesicle endocytosis, whereas UIMs are not. Strikingly, the efficacy of the rate of synaptic vesicle retrieval (the "endocytic capacity") was significantly decreased in the absence of Epsin1. Thus, Epsin1 is required for proper synaptic vesicle retrieval and modulates the endocytic capacity of synaptic vesicles.

Function and dysfunction of leucine-rich repeat kinase 2 (LRRK2): Parkinson's disease and beyond.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD). As such, functions and dysfunctions of LRRK2 in PD have been the subject of extensive investigation. In addition to PD, increasing evidence is suggesting that LRRK2 is associated with a wide range of diseases. Genome-wide association studies have implicated LRRK2 in Crohn's disease (CD) and leprosy, and the carriers with pathogenic mutations of LRRK2 show increased risk to develop particular types of cancer. LRRK2 mutations are rarely found in Alzheimer's disease (AD), but LRRK2 might play a part in tauopathies. The association of LRRK2 with the pathogenesis of apparently unrelated diseases remains enigmatic, but it might be related to the yet unknown diverse functions of LRRK2. Here, we reviewed current knowledge on the link between LRRK2 and several diseases, including PD, AD, CD, leprosy, and cancer, and discussed the possibility of targeting LRRK2 in such diseases.