Novel tankyrase inhibitors suppress TDP-43 aggregate formation.

Transactive response DNA-binding protein of 43 kDa (TDP-43) abnormally forms aggregates in certain subtypes of frontotemporal lobar degeneration (FTLD) and in amyotrophic lateral sclerosis (ALS). The pathological forms of TDP-43 have reported to be associated with poly(ADP-ribose) (PAR), which regulates the properties of these aggregates. A recent study has indicated that tankyrase, a member of the PAR polymerase (PARP) family, regulates pathological TDP-43 formation under conditions of stress, and tankyrase inhibitors suppress TDP-43 aggregate formation and cytotoxicity. Since we reported the development of tankyrase inhibitors that are more specific than conventional inhibitors, in this study, we examined their effects on the formation of TDP-43 aggregates in cultured cells. Time-lapse imaging showed that TDP-43 aggregates appeared in the nucleus within 30 min of treatment with sodium arsenite. Several tankyrase inhibitors suppressed the formation of aggregates and decreased the levels of the tankyrase protein. Immunohistochemical studies demonstrated that tankyrase was localized to neuronal cytoplasmic inclusions in the spinal cords of patients with ALS. Moreover, the tankyrase protein levels were significantly higher in the brains of patients with FTLD than in the brains of control subjects. These findings suggest that the inhibition of tankyrase activity protects against TDP-43 toxicity. Tankyrase inhibitors may be a potential treatment to suppress the progression of TDP-43 proteinopathies.

Autophagy mediators (FOXO1, SESN3 and TSC2) in Lewy body disease and aging.

Neurodegenerative disorders such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by impairment of autophagy. Cellular survival is dependent on efficient clearance of phosphorylated α-synuclein, which accumulates as fibrils in the neuronal cytoplasm as Lewy bodies (LBs). The forkhead box O 1 (FOXO1) is a member of the FOXO family that functions in various intracellular processes including regulation of autophagy. Transcriptional activation of FOXO1 has been reported to initiate autophagy by inhibiting the expression of Mechanistic Target of Rapamycin (mTOR), mediated by sestrin 3 (SESN3) and tuberous sclerosis complex 2 (TSC2). Although many autophagy-related proteins are known to be incorporated into LBs, no report has documented the involvement of these autophagy modulators (FOXO1, SESN3 and TSC2) in the pathogenesis of PD and DLB. In the present study, we performed immunostaining and Western blot analysis using the brains of normal controls and patients with PD and DLB in order to clarify the involvement of FOXO1, SESN3 and TSC2 in LBs. Our study demonstrated for the first time the presence of FOXO1, SESN3 and TSC2 in brainstem-type LBs. The expression levels of these proteins in the brain did not differ between the normal controls and patients with PD or DLB. We further utilized mice model to investigate the effect of α-synuclein overexpression on these proteins, and found that TSC2 was significantly increased in α-synuclein transgenic mice relative to wild type mice at 9 weeks of age, but not at 30 weeks of age. Together with expression data showing gradual increase of these molecules with age in wild type mice, these findings suggest that autophagy modulators are incorporated into LBs and that the expression of these proteins can be increased by various factors including aging.

YOD1 attenuates neurogenic proteotoxicity through its deubiquitinating activity.

Ubiquitination, a fundamental post-translational modification of intracellular proteins, is enzymatically reversed by deubiquitinase enzymes (deubiquitinases). >90 deubiquitinases have been identified. One of these enzymes, YOD1, possesses deubiquitinase activity and is similar to ovarian tumor domain-containing protein 1, which is associated with regulation of the endoplasmic reticulum (ER)-associated degradation pathway. Indeed, YOD1 is reported to be involved in the ER stress response induced by mislocalization of unfolded proteins in mammalian cells. However, it has remained unclear whether YOD1 is associated with pathophysiological conditions such as mitochondrial damage, impaired proteostasis, and neurodegeneration. We demonstrated that YOD1 possesses deubiquitinating activity and exhibits preference for K48- and K63-linked ubiquitin. Furthermore, YOD1 expression levels increased as a result of various stress conditions. We demonstrated that the neurogenic proteins that cause Huntington disease and Parkinson's disease induced upregulation of YOD1 level. We observed that YOD1 reduced disease cytotoxicity through efficient degradation of mutant proteins, whereas this activity was abolished by catalytically inactive YOD1. Additionally, YOD1 localized to Lewy bodies in Parkinson's disease patients. Collectively, these data suggest that the deubiquitinase YOD1 contributes to pathogenesis of neurodegenerative disease by decreasing ubiquitination of abnormal proteins and their subsequent degradation.

G protein-coupled receptor 26 immunoreactivity in intranuclear inclusions associated with polyglutamine and intranuclear inclusion body diseases.

G protein-coupled receptor 26 (GPR26) is one of the G-protein-coupled receptors (GPCRs), which comprise the largest family of membrane proteins and mediate most of the physiological responses to hormones, neurotransmitters and environmental stimulants. Although GPCRs are considered to play an important role in the pathophysiology of neurodegenerative disorders, it is uncertain whether GPR26 is involved in the pathogenesis of polyglutamine and intranuclear inclusion body diseases. We immunohistochemically examined the brain tissues of patients with four polyglutamine diseases (Huntington's disease, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxia types 1 and 3) and intranuclear inclusion body disease, and normal control subjects. In controls, anti-GPR26 antibody immunolabeled the neuronal cytoplasm in a diffuse granular pattern. Neuronal nuclear inclusions in polyglutamine diseases were immunopositive for GPR26. In intranuclear inclusion body disease, GPR26-positive nuclear inclusions were found in both neurons and glial cells. Marinesco bodies in aged control subjects were also positive for GPR26. Double immunofluorescence analysis revealed co-localization of GPR26 with polyglutamine or ubiquitin in these nuclear inclusions. These findings suggest that GPR26 may have a common role in the formation or degradation of intranuclear inclusions in several neurodegenerative diseases.

Sortilin-related receptor CNS expressed 2 (SorCS2) is localized to Bunina bodies in amyotrophic lateral sclerosis.

Sortilin-related receptor CNS expressed 2 (SorCS2) is one of the vacuolar protein sorting 10 family proteins (VPS10Ps) that have pleiotropic roles in protein trafficking and intracellular and intercellular signaling. Bunina bodies (BBs) are specifically detected in the lower motor neurons in patients with amyotrophic lateral sclerosis (ALS). BBs are immunolabeled with antibodies against cystatin C, transferrin and peripherin and are considered to originate from the endoplasmic reticulum, which is part of the protein sorting pathway. The present study investigated whether VPS10Ps are involved in the formation of BBs in ALS. We immunohistochemically examined the spinal cord from patients with ALS and control subjects using antibodies against VPS10Ps (sortilin, SorLA, SorCS1, SorCS2 and SorCS3). In normal controls, antibodies against VPS10Ps immunolabeled the cytoplasm of anterior horn cells in a fine granular pattern. In ALS, almost all BBs (95.1%) were strongly immunopositive for SorCS2, and immunoreativity for sortilin and SorLA was decreased in anterior horn cells. These findings suggest that VPS10Ps may be involved in the disease process of ALS.

Characteristic Gene Expression Profiles of Human Fibroblasts and Breast Cancer Cells in a Newly Developed Bilateral Coculture System.

The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment.

Proteomic and transcriptomic analyses of retinal pigment epithelial cells exposed to REF-1/TFPI-2.

PURPOSE: The authors previously reported a growth-promoting factor, REF-1/TFPI-2, that is specific to retinal pigment epithelial (RPE) cells. The purpose of this study was to determine the genes and proteins of human RPE cells that are altered by exposure to TFPI-2. METHODS: Human primary RPE cells were cultured with or without TFPI-2. Cell extracts and isolated RNA were subjected to proteomic and transcriptomic analyses, respectively. Proteins were separated by two-dimensional gel electrophoresis followed by gel staining and ion spray tandem mass spectrometry analyses. Transcriptomic analysis was performed using a DNA microarray to detect 27,868 gene expressions. RESULTS: Proteomic analysis revealed c-Myc binding proteins and ribosomal proteins L11 preferentially induced by TFPI-2 in human RPE cells. Transcriptomic analysis detected 10,773 of 33,096 probes in the TFPI-2 treated samples, whereas only 2186 probes were detected in the nontreated samples. Among the genes up-regulated by TFPI-2 at the protein level were c-myc, Mdm2, transcription factor E2F3, retinoblastoma binding protein, and the p21 gene, which is associated with the c-myc binding protein and ribosomal protein L11. CONCLUSIONS: The mechanisms by which TFPI-2 promotes the proliferation of RPE cells may be associated with augmented c-myc synthesis and the activation of E2F in the retinoblastoma protein (Rb)/E2F pathway at the G1 phase of the RPE cells. Activation of ribosomal protein L11 and the Mdm2 complex of the p53 pathway may be counterbalanced by the hyperproliferative conditions.