Therapeutic Effect of Padina arborescens Extract on a Cell System Model for Parkinson's Disease.

Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein are involved in the pathogenesis of Parkinson's disease. The activity of LRRK2 in microglial cells is associated with neuroinflammation, and LRRK2 inhibitors are crucial for alleviating this neuroinflammatory response. α-synuclein contributes to oxidative stress in the dopaminergic neuron and neuroinflammation through Toll-like receptors in microglia. In this study, we investigated the effect of the marine alga Padina arborescens on neuroinflammation by examining LRRK2 activation and the aggregation of α-synuclein. P. arborescens extract inhibits LRRK2 activity in vitro and decreases lipopolysaccharide (LPS)-induced LRRK2 upregulation in BV2, a mouse microglial cell line. Treatment with P. arborescens extract decreased tumor necrosis factor-α (TNF-α) gene expression by LPS through LRRK2 inhibition in BV2. It also attenuated TNF-α gene expression, inducible nitric oxide synthase, and the release of TNF-α and cellular nitric oxide in rat primary microglia. Furthermore, P. arborescens extract prevented rotenone (RTN)-induced oxidative stress in primary rat astrocytes and inhibited α-synuclein fibrilization in an in vitro assay using recombinant α-synuclein and in the differentiated human dopaminergic neuronal cell line SH-SY5Y (dSH). The extract increased lysosomal activity in dSH cells. In addition, P. arborescens extract slightly prolonged the lifespan of Caenorhabditis elegans, which was reduced by RTN treatment.

PUM1-TRAF3 fusion protein activates non-canonical NF-κB signaling via rescued NIK in biliary tract cancer.

Discovery and verification of diagnostic or therapeutic biomarkers for biliary tract cancer (BTC) is challenging owing to the low prevalence of the disease. Here, we identified and investigated the clinical impact of a fusion gene, Pumilio1-tumor necrosis factor receptor-associated factor 3 (PUM1-TRAF3), caused by 1;14 chromosomal translocation in BTC. PUM1-TRAF3 was initially identified in the RNA-sequencing of five BTC surgical tissues and confirmed by fluorescence in situ hybridization. Expression of the fusion gene was validated in an expanded cohort (5/55, 9.1%). Establishment and molecular assessment of PUM1-TRAF3 expressing BTC cells revealed that PUM1-TRAF3 activates non-canonical NF-κB signaling via NF-κB-inducing kinase (NIK). Abnormal TRAF3 activity, driven by competitive binding of PUM1-TRAF3 and TRAF3 to NIK, led to NIK rescue followed by P52/RelB nuclear translocation, all of which were reverted by an NIK inhibitor. The elevated expression of NIK and activated NF-κB signaling was observed in the PUM1-TRAF3-expressing regions of patient tissues. Expression of the PUM1-TRAF3 fusion was significantly correlated with strong NIK expression, which is associated with a poorer prognosis for patients with BTC. Overall, our study identifies a new fusion gene, PUM1-TRAF3, that activates NIK and non-canonical NF-κB signaling, which may be beneficial for developing precise treatment strategies for BTC.

Expression of G2019S LRRK2 in Rat Primary Astrocytes Mediates Neurotoxicity and Alters the Dopamine Synthesis Pathway in N27 Cells via Astrocytic Proinflammatory Cytokines and Neurotrophic Factors.

Astrocytes in the brain contribute to various essential functions, including maintenance of the neuronal framework, survival, communication, metabolic processes, and neurotransmitter levels. Leucine-rich repeat kinase 2 (LRRK2) is associated with the pathogenesis of Parkinson's disease (PD). LRRK2 is expressed in neurons, microglia, and astrocytes and plays diverse roles in these cell types. We aimed to determine the effects of mutant human G2019S-LRRK2 (GS-hLRRK2) in rat primary astrocytes (rASTROs). Transfection with GS-hLRRK2 significantly decreased cell viability compared to transfection with the vector and wild-type human LRRK2 (WT-hLRRK2). GS-hLRRK2 expression significantly reduced the levels of nerve growth factor and increased the levels of proinflammatory cytokines (interleukin-1ß and tumor necrosis factor α) compared to the vector and WT-hLRRK2 expression. Furthermore, GS-hLRRK2 expression in rASTROs promoted astrogliosis, which was characterized by increased expression of glial fibrillary acidic protein and vimentin. Treatment with the conditioned medium of G2019S LRRK2-expressing rASTROs decreased N27 cell viability compared to treatment with that of WT-hLRRK2-expressing rASTROs. Consequently, the regulation of the dopamine synthesis pathway was affected in N27 cells, thereby leading to altered levels of tyrosine hydroxylase, dopamine transporter, Nurr1, and dopamine release. Overall, the G2019S LRRK2 mutation disrupted astrocyte function, thereby aggravating PD progression.

SpatialSPM: statistical parametric mapping for the comparison of gene expression pattern images in multiple spatial transcriptomic datasets.

Spatial transcriptomic (ST) techniques help us understand the gene expression levels in specific parts of tissues and organs, providing insights into their biological functions. Even though ST dataset provides information on the gene expression and its location for each sample, it is challenging to compare spatial gene expression patterns across tissue samples with different shapes and coordinates. Here, we propose a method, SpatialSPM, that reconstructs ST data into multi-dimensional image matrices to ensure comparability across different samples through spatial registration process. We demonstrated the applicability of this method by kidney and mouse olfactory bulb datasets as well as mouse brain ST datasets to investigate and directly compare gene expression in a specific anatomical region of interest, pixel by pixel, across various biological statuses. Beyond traditional analyses, SpatialSPM is capable of generating statistical parametric maps, including T-scores and Pearson correlation coefficients. This feature enables the identification of specific regions exhibiting differentially expressed genes across tissue samples, enhancing the depth and specificity of ST studies. Our approach provides an efficient way to analyze ST datasets and may offer detailed insights into various biological conditions.

Immunological subtyping of salivary gland cancer identifies histological origin-specific tumor immune microenvironment.

Gene expression analysis enhances proper cancer subtyping, a better understanding of the molecular characteristics of cancer, and strategies for precision medicine. However, salivary gland cancer (SGC) subtyping remains largely unexplored because of its rarity and diverse histopathological and immunological characteristics. This study aimed to determine whether the histological origin and immunological characteristics of SGC subtypes are intrinsic tumor immunity factors. We performed immune profiling of 94 RNA-seq of SGC tissues and found that the SGCs that originated from the excretory duct (ED), such as the salivary duct and mucoepidermoid carcinomas, exhibit higher immunity than those from the intercalated duct (ID), such as the adenoid cystic and myoepithelial carcinomas, based on the computationally predicted immune score (p < 0.001), immune cell enrichment in the tumor immune microenvironment (TIME) (p < 0.001), T-cell receptor diversity (p < 0.001), and expression of signal I (major histocompatibility complex, MHC, p < 0.001) and signal II (co-stimulatory, p < 0.001 and co-inhibitory, p < 0.001) genes. Further analysis revealed that tolerogenic dendritic cell-induced dysfunctional T-cell populations and T-cell exclusion in the TIME are the major immune evasive mechanisms of the ED-and ID-derived SGCs, respectively.

Effects of liraglutide on depressive behavior in a mouse depression model and cognition in the probe trial of Morris water maze test.

BACKGROUND: We investigated the effects of liraglutide, a glucagon-like peptide-1 (GLP-1) agonist, on a depression-like phenotype in mice exposed to chronic unpredictable stress (CUS). Learning and memory were also assessed using the Morris water maze (MWM) test. METHODS: Liraglutide (0.3 mg/kg/day for 21 days) was administered to mice with or without exposure to CUS. After 21 days of CUS, the forced swim test (FST) was performed to assess its antidepressant effect. To evaluate cognitive function, liraglutide was administered to mice under stress-free conditions for 21 days, and then the MWM test was performed on 6 consecutive days. RESULTS: Chronic liraglutide treatment reduced FST immobility in mice with and without CUS. In the probe trial of the Morris water maze test, the search error rate was reduced and the time spent and path length in the target quadrant and the number of platform crossings were increased. LIMITATION: Additional animal model experiments and molecular level studies are needed to support the results obtained in this study. CONCLUSIONS: Liraglutide appears to exert antidepressant effects and could improve cognitive function. Based on these results, GLP-1 agonists could have potential as novel antidepressants.

Characterizing intrinsic molecular features of the immune subtypes of salivary mucoepidermoid carcinoma.

INTRODUCTION: Characterizing the tumor microenvironment (TME) and immune landscape of cancer has been a promising step towards discovering new therapeutic biomarkers and guiding precision medicine; however, its application in mucoepidermoid carcinoma (MEC) has been sparse. Here, we conducted a comprehensive study to understand the properties of the TME and immune profiles of MEC. METHOD: 20 patients with MEC were collected from Yonsei Head and Neck Cancer Centre, Yonsei University, South Korea. Total RNA sequencing was conducted to determine gene expression profiles. Bioinformatic and immunoinformatic analyses were applied to characterize the TME and identify immunophenotypic subgroups, and to investigate the molecular features that explain the distinct phenotypes. RESULTS: The MEC samples were subdivided into two groups, immune hot and immune cold, based on the heterogenous immune cell-infiltration and activation level. The immune-hot subgroup exhibited a higher level of immune activity, including T cell infiltration, cytolytic score, IFN-γ, antigen-presenting machinery, and immune modulator genes. Further characterizing molecular features of two subgroups, downregulation of lipid metabolic regulators, including MLXIPL and FASN, and the migration of chemokines and leukocytes were observed, respectively. And, Group-specific expression of immune checkpoint molecules, such as TIGIT, PD-L2, and CTLA-4, was observed in the immune-hot group, which can be exploited as a potential immunotherapeutic biomarker. CONCLUSIONS: Immunophenotypically heterogeneous MEC subgroups analysis has shown distinctive molecular characteristics and provided potential treatment options. These findings yield new insights into TME of MEC and may help next step to study this uncharted cancer.

Effects of Chronic LY341495 on Hippocampal mTORC1 Signaling in Mice with Chronic Unpredictable Stress-Induced Depression.

In several rodent models, acute administration of the metabotropic glutamate 2/3 (mGlu2/3) receptor antagonist LY341495 induced antidepressant-like effects via a mechanism of action similar to that of ketamine. However, the effects of chronic mGlu2/3 antagonism have not yet been explored. Therefore, we investigated the effects of chronic LY341495 treatment on the mechanistic target of rapamycin complex 1 (mTORC1) signaling and the levels of synaptic proteins in mice subjected to chronic unpredictable stress (CUS). LY341495 (1 mg/kg) was administered daily for 4 weeks to mice with and without CUS exposure. After the final treatment, the forced swimming test (FST) was used to assess antidepressant-like effects. The hippocampal levels of mTORC1-related proteins were derived by Western blotting. Chronic LY341495 treatment reversed the CUS-induced behavioral effects of FST. CUS significantly reduced the phosphorylation of mTORC1 and downstream effectors [eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP-1) and small ribosomal protein 6 (S6)], as well as the expression of synaptic proteins postsynaptic density-95 (PSD-95) and AMPA receptor subunit GluR1 (GluA1) in the hippocampus. However, chronic LY341495 treatment rescued these deficits. Our results suggest that the activation of hippocampal mTORC1 signaling is related to the antidepressant effect of chronic LY341495 treatment in an animal model of CUS-induced depression.

HLA-I-restricted CD8+ T cell immunity may accelerate tumorigenesis in conjunction with VHL inactivation.

CD8+ T cells recognize and kill tumor cells with HLA-I tumor antigens in early tumorigenesis, the efficiency of which differs according to antigen-recognition coverage, as shown in earlier tumor onset in HLA-I homozygosity. However, the universality of these associations remains unknown. Here, we assessed the tumor type and driver mutation specificity in the association between tumor onset age and HLA-I zygosity. Statistical analyses identified an unexpected negative relationship in tumors with VHL biallelic loss, wherein HLA-I heterozygosity was associated with earlier tumor onset, while all others showed either no or a positive association. Testing on an independent dataset reproduced the VHL-dependent acceleration of tumor onset in the HLA-I heterozygous group, confirming the association. Further speculation proposed VEGF-A-mediated T cell exhaustion under VHL inactivation as a potential mechanism. Our findings suggest that CD8+ T cell immunity in early tumor suppression can be conditional to the genetic status of tumors and may even lead to adverse consequences.

Differential expression of gene co-expression networks related to the mTOR signaling pathway in bipolar disorder.

Bipolar disorder (BPD) is a severe mental illness characterized by episodes of depression and mania. To investigate the molecular mechanisms underlying the pathophysiology of bipolar disorder, we performed transcriptome studies using RNA-seq data from the prefrontal cortex (PFC) of individuals with BPD and matched controls, as well as data from cell culture and animal model studies. We found 879 differentially expressed genes that were also replicated in an independent cohort of post-mortem samples. Genes involving the mechanistic target of rapamycine (mTOR) pathway were down-regulated, while genes interrelated with the mTOR pathway such as Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway were up-regulated. Gene co-expression network analyses identified a module related to the mTOR pathway that was up-regulated in BPD and also enriched for markers of endothelial cells. We also found a down-regulated co-expression module enriched for genes involved in mTOR signalling and in mTOR related pathways and enriched with neuronal markers. The mTOR related modules were also replicated in the independent cohort of samples. To investigate whether the expression of the modules related to mTOR signalling pathway could be differentially regulated in different cell types we performed comparative network analyses in experimental models. We found both up-regulated modules in the PFC significantly overlapped with an up-regulated module in the brain endothelial cells from mice treated with lipopolysaccharides (LPS) and mTOR related pathways such as JAK-STAT, PI3K-Akt and ribosome were enriched in the common genes. In addition, the down-regulated module in the PFC significantly overlapped with a down-regulated module from neurons treated with the mTOR inhibitor, Torin1 and mTOR signalling, autophagy, and synaptic vesicle cycles were significantly enriched in the common genes. These results suggest that co-expression networks related to mTOR signalling pathways may be up- or down-regulated in different cell types in the PFC of BPD. These results provide novel insights into the molecular mechanisms underlying the pathophysiology of BPD.

Neuromolecular Etiology of Bipolar Disorder: Possible Therapeutic Targets of Mood Stabilizers.

Bipolar disorder is a mental illness that causes extreme mood swings and has a chronic course. However, the mechanism by which mood episodes with completely opposite characteristics appear repeatedly, or a mixture of symptoms appears, in patients with bipolar disorder remains unknown. Therefore, mood stabilizers are indicated only for single mood episodes, such as manic episodes and depressive episodes, and no true mood-stabilizing drugs effective for treating both manic and depressive episodes currently exist. Therefore, in this review, therapeutic targets that facilitate the development of mood stabilizers were examined by reviewing the current understanding of the neuromolecular etiology of bipolar disorder.

Tumor microenvironment-aware, single-transcriptome prediction of microsatellite instability in colorectal cancer using meta-analysis.

Detecting microsatellite instability (MSI) in colorectal cancers (CRCs) is essential because it is the determinant of treatment strategies, including immunotherapy and chemotherapy. Yet, no attempt has been made to exploit transcriptomic profile and tumor microenvironment (TME) of it to unveil MSI status in CRC. Hence, we developed a novel TME-aware, single-transcriptome predictor of MSI for CRC, called MAP (Microsatellite instability Absolute single sample Predictor). MAP was developed utilizing recursive feature elimination-random forest with 466 CRC samples from The Cancer Genome Atlas, and its performance was validated in independent cohorts, including 1118 samples. MAP showed robustness and predictive power in predicting MSI status in CRC. Additional advantages for MAP were demonstrated through comparative analysis with existing MSI classifier and other cancer types. Our novel approach will provide access to untouched vast amounts of publicly available transcriptomic data and widen the door for MSI CRC research and be useful for gaining insights to help with translational medicine.

Comprehensive clinicopathologic, molecular, and immunologic characterization of colorectal carcinomas with loss of three intestinal markers, CDX2, SATB2, and KRT20.

Caudal-type homeobox 2 (CDX2), special AT-rich sequence-binding protein 2 (SATB2), and keratin 20 (KRT20) are frequently used as intestinal epithelium-specific markers in immunohistochemical studies. However, subsets of colorectal carcinomas (CRCs) show loss of these markers. We analyzed The Cancer Genome Atlas data to explore molecular correlates of CDX2, SATB2, and KRT20 genes in 390 CRCs. The decreased mRNA expression of each of the three genes commonly correlated with microsatellite instability-high (MSI-H), CpG island methylator phenotype-high (CIMP-H), BRAF/RNF43 mutations, consensus molecular subtype 1, and high tumor mutational burden. The downregulation of CDX2 or SATB2 was dependent on both MSI-H and CIMP-H, whereas that of KRT20 was more dependent on MSI-H than on CIMP-H. Next, we evaluated the immunohistochemical expression of CDX2, SATB2, and KRT20 in 436 primary CRCs. In contrast to RNA-level expression, decreased expression of CDX2 and SATB2 was more dependent on CIMP-H than on MSI-H. However, consistent with RNA-level expression, decreased expression of KRT20 was more dependent on MSI-H than on CIMP-H. CIMP-H and lymphatic invasion were consistently associated with both CDX2 loss and SATB2 loss in CRCs, regardless of MSI status. In microsatellite stable CRCs, CDX2 loss correlated with BRAF mutation, whereas SATB2 loss was associated with KRAS mutations and decreased T-cell infiltration. Cases with concurrent loss of all three markers were found exclusively in MLH1-methylated MSI-H/CIMP-H CRCs. In conclusion, MSI-H and/or CIMP-H are major common correlates of decreased CDX2/SATB2/KRT20 expression in CRCs, but the specific features associated with the loss of each marker are different in CRCs.

Genomic and transcriptomic characterization of heterogeneous immune subgroups of microsatellite instability-high colorectal cancers.

BACKGROUND: Colorectal cancers (CRCs) with microsatellite instability-high (MSI-H) are hypermutated tumors and are generally regarded as immunogenic. However, their heterogeneous immune responses and underlying molecular characteristics remain largely unexplained. METHODS: We conducted a retrospective analysis of 73 primary MSI-H CRC tissues to characterize heterogeneous immune subgroups. Based on combined tumor-infiltrating lymphocyte (TIL) immunoscore and tertiary lymphoid structure (TLS) activity, MSI-H CRCs were classified into immune-high, immune-intermediate, and immune-low subgroups. Of these, the immune-high and immune-low subgroups were further analyzed using whole-exome and transcriptome sequencing. RESULTS: We found considerable variations in immune parameters between MSI-H CRCs, and immune subgrouping of MSI-H CRCs was performed accordingly. The TIL densities and TLS activities of immune-low MSI-H CRCs were comparable to those of an immune-low or immune-intermediate subgroup of microsatellite-stable CRCs. There were remarkable differences between immune-high and immune-low MSI-H CRCs, including their pathological features (medullary vs mucinous), genomic alterations (tyrosine kinase fusions vs KRAS mutations), and activated signaling pathways (immune-related vs Wnt and Notch signaling), whereas no significant differences were found in tumor mutational burden (TMB) and neoantigen load. The immune-low MSI-H CRCs were subdivided by the consensus molecular subtype (CMS1 vs CMS3) with different gene expression signatures (mesenchymal/stem-like vs epithelial/goblet-like), suggesting distinct immune evasion mechanisms. Angiogenesis and CD200 were identified as potential therapeutic targets in immune-low CMS1 and CMS3 MSI-H CRCs, respectively. CONCLUSIONS: MSI-H CRCs are immunologically heterogeneous, regardless of TMB. The unusual immune-low MSI-H CRCs are characterized by mucinous histology, KRAS mutations, and Wnt/Notch activation, and can be further divided into distinct gene expression subtypes, including CMS4-like CMS1 and CMS3. Our data provide novel insights into precise immunotherapeutic strategies for subtypes of MSI-H tumors.

Early life stress induces age-dependent epigenetic changes in p11 gene expression in male mice.

Early life stress (ELS) causes long-lasting changes in gene expression through epigenetic mechanisms. However, little is known about the effects of ELS in adulthood, specifically across different age groups. In this study, the epigenetic modifications of p11 expression in adult mice subjected to ELS were investigated in different stages of adulthood. Pups experienced maternal separation (MS) for 3 h daily from postnatal day 1 to 21. At young and middle adulthood, behavioral test, hippocampal p11 expression levels, and levels of histone acetylation and methylation and DNA methylation at the hippocampal p11 promoter were measured. Middle-aged, but not young adult, MS mice exhibited increased immobility time in the forced swimming test. Concurrent with reduced hippocampal p11 levels, mice in both age groups showed a decrease in histone acetylation (AcH3) and permissive histone methylation (H3K4me3) at the p11 promoter, as well as an increase in repressive histone methylation (H3K27me3). Moreover, our results showed that the expression, AcH3 and H3Kme3 levels of p11 gene in response to MS were reduced with age. DNA methylation analysis of the p11 promoter revealed increased CpG methylation in middle-aged MS mice only. The results highlight the age-dependent deleterious effects of ELS on the epigenetic modifications of p11 transcription.

NTRK oncogenic fusions are exclusively associated with the serrated neoplasia pathway in the colorectum and begin to occur in sessile serrated lesions.

Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are emerging tissue-agnostic drug targets in malignancies including colorectal carcinomas (CRCs), but their detailed landscape in the context of various colorectal carcinogenesis pathways remains to be investigated. In this study, pan-tropomyosin receptor kinase (TRK) protein expression was assessed by immunohistochemistry (IHC) in retrospectively collected colorectal epithelial tumor tissues, including 441 CRCs [133 microsatellite instability-high (MSI-high) and 308 microsatellite stable (MSS)] and 595 premalignant colorectal lesions (330 serrated lesions and 265 conventional adenomas). TRK-positive cases were then subjected to next-generation sequencing and/or fluorescence in situ hybridization to confirm NTRK rearrangements. TRK IHC positivity was not observed in any of the MSS CRCs, conventional adenomas, traditional serrated adenomas, or hyperplastic polyps, whereas TRK positivity was observed in 11 of 58 (19%) MLH1-methylated MSI-high CRCs, 4 of 23 (17%) sessile serrated lesions with dysplasia (SSLDs), and 5 of 132 (4%) sessile serrated lesions (SSLs). The 11 TRK-positive MSI-high CRCs commonly harbored CpG island methylator phenotype-high (CIMP-high), MLH1 methylation, BRAF/KRAS wild-type, and NTRK1 or NTRK3 fusion (TPM3-NTRK1, TPR-NTRK1, LMNA-NTRK1, SFPQ-NTRK1, ETV6-NTRK3, or EML4-NTRK3). Both NTRK1 or NTRK3 rearrangement and BRAF/KRAS wild-type were detected in all nine TRK-positive SSL(D)s, seven of which demonstrated MSS and/or CIMP-low. TRK expression was selectively observed in distorted serrated crypts within SSLs and was occasionally localized at the base of serrated crypts. NTRK fusions were detected only in SSLs of patients aged ≥50 years, whereas BRAF mutation was found in younger age-onset SSLs. In conclusion, NTRK-rearranged colorectal tumors develop exclusively through the serrated neoplasia pathway and can be initiated from non-dysplastic SSLs without BRAF/KRAS mutations prior to full occurrence of MSI-high/CIMP-high. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

LRRK2 Kinase Inhibitor Rejuvenates Oxidative Stress-Induced Cellular Senescence in Neuronal Cells.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) plays a critical role in the pathogenesis of Parkinson's disease (PD). Aging is the most critical risk factor for the progression of PD. The correlation between aging and cellular senescence has been established. Cellular senescence is correlated with the dysregulation of the proteolytic pathway and mitochondrial dysfunction, which are also associated with the aggregation of α-synuclein (α-syn). METHODS: Human dopaminergic neuron-like cells (differentiated SH-SY5Y cells) were treated with rotenone in the presence or absence of the LRRK2 kinase inhibitor GSK2578215A (GSK-KI) for 48 h. The markers of cellular senescence, including p53, p21Waf1/Cip1 (p21), ß-galactosidase (ß-gal), Rb phosphorylation, senescence-associated (SA) ß-gal activity, and lysosomal activity, were examined. The dSH cells and rat primary cortical neurons were treated with α-syn fibrils 30 min before treatment with rotenone in the presence or absence of GSK-KI for 48 h. Mice were intraperitoneally injected with rotenone and MLi-2 (LRRK2 kinase inhibitor) once every two days for two weeks. RESULTS: Rotenone upregulated LRRK2 phosphorylation and ß-gal levels through the activation of the p53-p21 signaling axis and downregulated Rb phosphorylation. Additionally, rotenone upregulated SA ß-gal activity, reactive oxygen species levels, and LRRK2 phosphorylation and inhibited lysosome activity. Rotenone-induced LRRK2 upregulation impaired the clearance of α-syn fibrils. Treatment with LRRK2 inhibitor mitigated rotenone-induced cellular senescence and α-syn accumulation. CONCLUSIONS: Rotenone-induced upregulation of LRRK2 kinase activity promoted cellular senescence, which enhanced α-syn accumulation. However, the administration of an LRRK2 kinase inhibitor rejuvenated rotenone-induced cellular senescence.

Ciliogenesis is Not Directly Regulated by LRRK2 Kinase Activity in Neurons.

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of familial Parkinson's disease (PD). The increase in LRRK2 kinase activity observed in the pathogenic G2019S mutation is important for PD development. Several studies have reported that increased LRRK2 kinase activity and treatment with LRRK2 kinase inhibitors decreased and increased ciliogenesis, respectively, in mouse embryonic fibroblasts (MEFs) and retinal pigment epithelium (RPE) cells. In contrast, treatment of SH-SY5Y dopaminergic neuronal cells with PD-causing chemicals increased ciliogenesis. Because these reports were somewhat contradictory, we tested the effect of LRRK2 kinase activity on ciliogenesis in neurons. In SH-SY5Y cells, LRRK2 inhibitor treatment slightly increased ciliogenesis, but serum starvation showed no increase. In rat primary neurons, LRRK2 inhibitor treatment repeatedly showed no significant change. Little difference was observed between primary cortical neurons prepared from wild-type (WT) and G2019S+/- mice. However, a significant increase in ciliogenesis was observed in G2019S+/- compared to WT human fibroblasts, and this pattern was maintained in neural stem cells (NSCs) differentiated from the induced pluripotent stem cells (iPSCs) prepared from the same WT/G2019S fibroblast pair. NSCs differentiated from G2019S and its gene-corrected WT counterpart iPSCs were also used to test ciliogenesis in an isogenic background. The results showed no significant difference between WT and G2019S regardless of kinase inhibitor treatment and B27-deprivation-mimicking serum starvation. These results suggest that LRRK2 kinase activity may be not a direct regulator of ciliogenesis and ciliogenesis varies depending upon the cell type or genetic background.

Expression of transduced nucleolin promotes the clearance of accumulated α-synuclein in rodent cells and animal model.

Alpha-synuclein (αSyn) is a major component of Lewy bodies, which are a known pathogenic marker of Parkinson's disease (PD). The dysfunction of protein degradation machinery causes αSyn accumulation. The reinforcement of αSyn degradation is a potential therapeutic target for PD because accumulated αSyn is responsible for the pathogenesis of PD. Nucleolin (NCL) is essential in the formation of the nucleolar structure. The function of NCL is correlated with oxidative stress-mediated cell death. A previous study demonstrated that NCL overexpression alleviated rotenone-induced neurotoxic effects, whereas knockdown of NCL had the opposite effect. These results suggest that NCL malfunction would exacerbate PD pathology. Thus, it was hypothesized that the introduction of ectopic NCL could rescue α-synucleinopathy in PD. This study investigated whether the ectopic expression of NCL facilitates αSyn clearance. Ectopic expression of NCL was accomplished via the transfection of green fluorescent protein (GFP) or GFP-NCL in mouse embryonic fibroblasts (MEF) or transduction of GFP or GFP-NCL using lentivirus in rat primary cortical neurons and mouse substantia nigra. NCL overexpression enhanced the clearance of accumulated or aggregated αSyn in MEFs and rat primary cortical neurons. The activity of the autophagy-lysosome pathway was enhanced by NCL expression. NCL transduction in the substantia nigra, which was co-injected with αSyn fibrils, rescued PD manifestation. The elevation of NCL levels may reflect a therapeutic strategy for α-synucleinopathy in PD.