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author:("Tanaka, kenji")
1.  Specialized proteasome subunits play an essential role in thymic selection of CD8+ T cells 
Nature immunology  2016;17(8):938-945.
The cells that stimulate positive selection express different specialized proteasome β-subunits than all other cells, including those involved in negative selection. Mice that lack all four specialized proteasome β-subunits, and therefore express only constitutive proteasomes in all cells, had a profound defect in the generation of CD8+ T cells. While a defect in positive selection would reflect an inability to generate the appropriate positively selecting peptides, a block at negative selection would point to the potential need to switch peptides between positive and negative selection to avoid the two processes often cancelling each other out. We found that the block in T cell development occurred around the checkpoints of positive and, surprisingly, also negative selection.
doi:10.1038/ni.3480
PMCID: PMC4955723  PMID: 27294792
2.  The Neural System of Postdecision Evaluation in Rostral Frontal Cortex during Problem-solving Tasks 
eNeuro  2016;3(4):ENEURO.0188-16.2016.
Visual Abstract
Little attention has been paid to the postdecision processing in fMRI studies with task paradigms in which there was no explicit feedback. Although late-onset BOLD responses were previously observed in the lateral frontopolar cortex after the familiar-novel decision on visually presented words, the nature of neural activations that caused the late-onset BOLD responses remained elusive. We here found, in human experts conducting complicated problem-solving tasks in their expertise domain, that the rostral frontal cortex, including the lateral frontopolar cortex, along with the anterior inferior parietal lobule, was activated only during the postdecision period, although there was no feedback. That is, these areas showed late-onset BOLD responses, and fitting of the BOLD responses with different models indicates that they were caused by neural activations that occurred after the decision. However, there was no response after performing a sensory-motor control task, and the magnitude of postdecision activations was correlated with the degree of uncertainty about the preceding decision, which suggests that the postdecision neural activations were associated with the preceding decision procedure. Furthermore, the same set of areas was more strongly activated when the subject explicitly rethought the preceding problem. These results suggest that the rostral frontal cortex, together with anterior inferior parietal lobule, comprises a network for uncertainty monitoring and exploration of alternative resolutions in postdecision evaluation. The present results thus introduce a new aspect of the functional gradient along the rostrocaudal axis in the frontal cortex.
doi:10.1523/ENEURO.0188-16.2016
PMCID: PMC5002985  PMID: 27595134
dorsal anterior cingulate cortex; expert; fMRI; frontopolar cortex; metacognition; shogi
3.  The PARK2/Parkin receptor on damaged mitochondria revisited—uncovering the role of phosphorylated ubiquitin chains 
Autophagy  2015;11(9):1700-1701.
Phosphorylated ubiquitin produced by PINK1 kinase functions as a PARK2/Parkin activator by derepressing intramolecular autoinhibition of PARK2 E3 activity. Unexpectedly, we revealed that phosphorylated polyubiquitin chain also functions in the PARK2 recruitment process as a PARK2 receptor. Phosphorylated ubiquitin enables us to comprehensively understand how PINK1 and PARK2 catalyzes (phospho-)ubiquitination of depolarized mitochondria and subsequent mitophagy.
doi:10.1080/15548627.2015.1071760
PMCID: PMC4590609  PMID: 26213095
mitochondria; PARK2/Parkin; PARK2 receptor; phosphorylated ubiquitin; PINK1
4.  p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming 
Nature Communications  2016;7:12030.
p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.
Dysregulation of p62 has been implicated in tumorigenesis. Here, the authors show that p62 promotes hepatocellular carcinoma by reprogramming glucose and glutamine metabolism through Nrf2 and present a novel compound that can inhibit p62 action thus sensitizing cancer cells to chemotherapy.
doi:10.1038/ncomms12030
PMCID: PMC4931237  PMID: 27345495
5.  Cross-National Variation in Glycemic Control and Diabetes-Related Distress Among East Asian Patients Using Insulin: Results from the MOSAIc Study 
Diabetes Therapy  2016;7(2):349-360.
Introduction
Guidelines recommend insulin progression for patients with type 2 diabetes (T2D) with inadequate glycemic control. The Multinational Observational Study Assessing Insulin use (MOSAIc [ClinicalTrials.gov identifier, NCT01400971]) study is a 2-year observational study, investigating factors that influence insulin progression in T2D patients. In this first of two reports, we describe baseline clinical and psychosocial characteristics of Chinese, Japanese, and South Korean patients who participated in MOSAIc. Insulin treatment, factors affecting progression, and outcomes will be reported separately.
Methods
Patients with T2D using insulin for ≥3 months were eligible. Baseline demographic, clinical, and psychosocial data were collected from patients. Quality of life instruments, including the Diabetes Distress Scale (DDS), were used to assess patient’s concerns about disease management, support, and emotional burden. The association between the DDS and the selected covariates was also assessed.
Results
A total of 373 patients in China, 157 in Japan, and 141 in South Korea were enrolled from July 2011 to July 2013. Mean ± standard deviation duration (years) of T2D differed across countries (China 11.4 ± 7.5; Japan 13.8 ± 8.7; South Korea 15.7 ± 8.8; P < 0.0001). Japanese patients used more noninsulin anti-hyperglycemic agents than did Chinese or South Korean patients (P < 0.0001). Exclusive use of basal insulin was most common in Japan and South Korea compared with China, whereas approximately 66.8% of Chinese patients used mixed insulin. Covariates associated with the DDS were younger age [P = 0.044 (Japan)], higher incidence of monthly hypoglycemia [P = 0.036 [China]; P = 0.021 (South Korea)], and male gender [P = 0.037 (South Korea)].
Conclusions
There were significant differences amongst East Asian patients with T2D treated with insulin, including in quality of life scores. Results from the MOSAIc longitudinal analyses will further investigate trends of insulin intensification and barriers to insulin progression.
Funding
Eli Lilly and Company.
doi:10.1007/s13300-016-0178-7
PMCID: PMC4900985  PMID: 27255328
Asia; Diabetes Mellitus; Hypoglycemia; Insulin; Patient-reported outcomes; Type 2
6.  TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8+ T cells 
Nature immunology  2015;16(10):1069-1076.
In the thymus, low-affinity T cell antigen receptor (TCR) engagement facilitates positive selection of a useful T cell repertoire. Here we report that TCR responsiveness of mature CD8+ T cells is fine-tuned by their affinity for positively selecting peptides in the thymus and that optimal TCR responsiveness requires positive selection on MHC class I-associated peptides produced by the thymoproteasome, which is specifically expressed in the thymic cortical epithelium. Thymoproteasome-independent positive selection of monoclonal CD8+ T cells results in aberrant TCR responsiveness, homeostatic maintenance, and immune responses to infection. These results demonstrate a novel aspect of positive selection, in which TCR affinity for positively selecting peptides produced by thymic epithelium determines the subsequent antigen responsiveness of mature CD8+ T cells in the periphery.
doi:10.1038/ni.3237
PMCID: PMC4810782  PMID: 26301566
7.  Factors Influencing Changes in Hemoglobin A1c and Body Weight During Treatment of Type 2 Diabetes With Ipragliflozin: Interim Analysis of the ASSIGN-K Study 
Background
Ipragliflozin is a selective sodium glucose co-transporter 2 (SGLT2) inhibitor that blocks glucose reabsorption in the proximal tubules. SGLT2 inhibitors are expected to be effective in patients with insulin resistance and obesity, but it is important to select treatment according to patient background factors that minimizes the risk of adverse events. There have been a limited number of investigations into the relationship between the clinical efficacy (reducing hemoglobin A1c (HbA1c) and body weight (BW)) or safety of SGLT2 inhibitors and patient characteristics.
Methods
ASSIGN-K is an investigator-initiated, multicenter, prospective observational study examining the efficacy and safety of ipragliflozin (50 - 100 mg/day for 52 weeks) in Japanese patients with type 2 diabetes mellitus (T2DM) who had inadequate glycemic control with HbA1c ≥ 6.0% (National Glycohemoglobin Standardization Program) despite diet and exercise therapy or diet and exercise plus antidiabetic drug therapy. We conducted an interim analysis of the relationship between changes in HbA1c or BW and characteristics in patients who had been on treatment for more than 12 weeks.
Results
In 257 patients completing 12 weeks of treatment, HbA1c decreased significantly from 8.23% to 7.55% (-0.68%, P < 0.01). The change in HbA1c after 12 weeks was -0.17%, -0.33%, and -1.16% when baseline HbA1c was < 7%, 7% to < 8%, and ≥ 8%, respectively (P < 0.05, P < 0.01, and P < 0.01, respectively), and -1.30%, -0.62%, and -0.62% when baseline body mass index (BMI) was < 25, 25 to < 30, and ≥ 30, respectively (all P < 0.01). Stratified analysis showed that age, gender, or BMI did not have a significant influence on the improvement in HbA1c. Multiple regression analysis showed that reduction in HbA1c was greater as baseline HbA1c increased and the duration of diabetes decreased. A higher baseline HbA1c was associated with less weight loss.
Conclusions
Ipragliflozin significantly improved HbA1c in patients with T2DM. HbA1c improved more when baseline HbA1c was higher and the duration of diabetes was shorter, suggesting that current treatment policies for diabetes could be re-examined.
doi:10.14740/jocmr2492w
PMCID: PMC4817576  PMID: 27081422
Type 2 diabetes; Ipragliflozin; Selective sodium glucose co-transporter 2 inhibitor; Hemoglobin A1c; Body weight; Patient characteristics; Interim analysis
9.  Efficacy and Safety of Ipragliflozin in Japanese Patients With Type 2 Diabetes: Interim Outcome of the ASSIGN-K Study 
Background
Ipragliflozin is a sodium-glucose co-transporter 2 inhibitor that can improve glycemic control and reduce body weight and blood pressure in patients with type 2 diabetes mellitus (T2DM). We evaluated the efficacy and safety of ipragliflozin in the real-world clinical setting, with a focus on the changes of body composition up to 3 months of treatment.
Methods
This was a prospective multicenter interventional trial. We investigated changes of the blood pressure, body composition, blood glucose, hemoglobin A1c (HbA1c), ketone bodies, lipids, and insulin after treatment with ipragliflozin (50 - 100 mg/day) for 12 weeks in Japanese patients with T2DM who showed poor glycemic control despite receiving diet and exercise therapy with or without oral antidiabetic drugs for more than 12 weeks.
Results
Two hundred and fifty-seven subjects were included in the efficacy analysis up to 12 weeks of treatment and 301 subjects were included in the safety analysis. From baseline to 12 weeks, HbA1c showed a change of -0.68% (95% confidence interval (CI): -0.83, -0.53) and fasting blood glucose showed a change of -23.9 mg/dL (95% CI: -30.5, -17.2), with both parameters displaying a significant reduction (P < 0.001). The difference of body weight from baseline was -1.82 kg (95% CI: -2.14, -1.50), and it also showed significant reduction (P < 0.001). Analysis of body composition revealed that body fat changed by -1.46 kg (95% CI: -1.79, -1.14, P < 0.001) and body water changed by -0.37 kg (95% CI: -0.60, -0.14, P < 0.01). Laboratory tests demonstrated improvement of liver function and the lipid profile. Adverse events (AEs) occurred in 22.6% of the subjects, with frequent events being vulvovaginal candidiasis in 2.7% and cystitis in 2.0%. Serious AEs occurred in three subjects.
Conclusions
In patients with T2DM, ipragliflozin improved glycemic control after 1 month of treatment and caused weight loss by reducing body fat more than body water.
doi:10.14740/jocmr2417w
PMCID: PMC4701067  PMID: 26767080
Body composition; Type 2 diabetes mellitus; Sodium-glucose co-transporter 2 inhibitor; Ipragliflozin; Antidiabetic agent
10.  Phosphorylated ubiquitin chain is the genuine Parkin receptor 
The Journal of Cell Biology  2015;209(1):111-128.
PINK1-phosphorylated ubiquitin chain is the genuine Parkin receptor that recruits Parkin to depolarized mitochondria.
PINK1 selectively recruits Parkin to depolarized mitochondria for quarantine and removal of damaged mitochondria via ubiquitylation. Dysfunction of this process predisposes development of familial recessive Parkinson’s disease. Although various models for the recruitment process have been proposed, none of them adequately explain the accumulated data, and thus the molecular basis for PINK1 recruitment of Parkin remains to be fully elucidated. In this study, we show that a linear ubiquitin chain of phosphomimetic tetra-ubiquitin(S65D) recruits Parkin to energized mitochondria in the absence of PINK1, whereas a wild-type tetra-ubiquitin chain does not. Under more physiologically relevant conditions, a lysosomal phosphorylated polyubiquitin chain recruited phosphomimetic Parkin to the lysosome. A cellular ubiquitin replacement system confirmed that ubiquitin phosphorylation is indeed essential for Parkin translocation. Furthermore, physical interactions between phosphomimetic Parkin and phosphorylated polyubiquitin chain were detected by immunoprecipitation from cells and in vitro reconstitution using recombinant proteins. We thus propose that the phosphorylated ubiquitin chain functions as the genuine Parkin receptor for recruitment to depolarized mitochondria.
doi:10.1083/jcb.201410050
PMCID: PMC4395490  PMID: 25847540
11.  The Structural Differences between a Glycoprotein Specific F-Box Protein Fbs1 and Its Homologous Protein FBG3 
PLoS ONE  2015;10(10):e0140366.
The Skp1-Cul1-F-box protein (SCF) complex catalyzes protein ubiquitination in diverse cellular processes and is one of the best-characterized ubiquitin ligases. F-box proteins determine the substrate specificities of SCF ubiquitin ligases. Among these, Fbs1/FBG1/FBXO2, Fbs2/FBG2/FBXO6, and Fbs3/FBG5/FBXO27 recognize the N-glycans of glycoproteins, whereas FBG3/FBXO44 has no sugar-binding activity, despite the high sequence homology and conservation of the residues necessary for oligosaccharide binding between Fbs1–3 and FBG3. Here we determined the crystal structure of the Skp1–FBG3 complex at a resolution of 2.6 Å. The substrate-binding domain of FBG3 is composed of a 10-stranded antiparallel β-sandwich with three helices. Although the overall structure of FBG3 is similar to that of Fbs1, the residues that form the Fbs1 carbohydrate-binding pocket failed to be superposed with the corresponding residues of FBG3. Structure-based mutational analysis shows that distinct hydrogen bond networks of four FBG3 loops, i.e., β2-β3, β5-β6, β7-β8, and β9-β10, prevent the formation of the carbohydrate-binding pocket shown in Fbs1.
doi:10.1371/journal.pone.0140366
PMCID: PMC4603797  PMID: 26460611
12.  Conserved Mode of Interaction between Yeast Bro1 Family V Domains and YP(X)nL Motif-Containing Target Proteins 
Eukaryotic Cell  2015;14(10):976-982.
Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X)nL (n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X)nL motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X)nL-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X)nL motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity.
doi:10.1128/EC.00091-15
PMCID: PMC4588623  PMID: 26150415
13.  Hue Selectivity in Human Visual Cortex Revealed by Functional Magnetic Resonance Imaging 
Cerebral Cortex (New York, NY)  2015;25(12):4869-4884.
The variability of color-selective neurons in human visual cortex is considered more diverse than cone-opponent mechanisms. We addressed this issue by deriving histograms of hue-selective voxels measured using fMRI with a novel stimulation paradigm, where the stimulus hue changed continuously. Despite the large between-subject difference in hue-selective histograms, individual voxels exhibited selectivity for intermediate hues, such as purple, cyan, and orange, in addition to those along cone-opponent axes. In order to rule the possibility out that the selectivity for intermediate hues emerged through spatial summation of activities of neurons selectively responding to cone-opponent signals, we further tested hue-selective adaptations in intermediate directions of cone-opponent axes, by measuring responses to 4 diagonal hues during concurrent adaptation to 1 of the 4 hues. The selective and unidirectional reduction in response to the adapted hue lends supports to our argument that cortical neurons respond selectively to intermediate hues.
doi:10.1093/cercor/bhv198
PMCID: PMC4635924  PMID: 26423093
color vision; fMRI adaptation; functional MRI; hue selectivity; human visual cortex; population histogram
14.  Thymoproteasomes produce unique peptide motifs for positive selection of CD8+ T cells 
Nature Communications  2015;6:7484.
Positive selection in the thymus provides low-affinity T-cell receptor (TCR) engagement to support the development of potentially useful self-major histocompatibility complex class I (MHC-I)-restricted T cells. Optimal positive selection of CD8+ T cells requires cortical thymic epithelial cells that express β5t-containing thymoproteasomes (tCPs). However, how tCPs govern positive selection is unclear. Here we show that the tCPs produce unique cleavage motifs in digested peptides and in MHC-I-associated peptides. Interestingly, MHC-I-associated peptides carrying these tCP-dependent motifs are enriched with low-affinity TCR ligands that efficiently induce the positive selection of functionally competent CD8+ T cells in antigen-specific TCR-transgenic models. These results suggest that tCPs contribute to the positive selection of CD8+ T cells by preferentially producing low-affinity TCR ligand peptides.
Proteasomes digest intracellular proteins into peptides that are then presented to lymphocytes as antigens. Here the authors show that a thymic epithelium-specific proteasome subunit cuts model proteins in a pattern favouring their weak binding to T cell receptor, and thus T cell positive selection.
doi:10.1038/ncomms8484
PMCID: PMC4557289  PMID: 26099460
15.  Factors Predicting Therapeutic Efficacy of Combination Treatment With Sitagliptin and Insulin in Type 2 Diabetic Patients: The ASSIST-K Study 
Background
It is unclear whether dipeptidyl peptidase-4 inhibitors decrease hemoglobin A1c (HbA1c) in a glucose-dependent manner in patients on insulin therapy who have impaired insulin secretion. This study investigated factors influencing the efficacy of sitagliptin when used concomitantly with insulin to treat type 2 diabetes mellitus (T2DM) in the real-world setting.
Methods
A retrospective study was conducted of 1,004 T2DM patients at 36 Japanese clinics associated with the Diabetes Task Force of the Kanagawa Physicians Association. Eligible patients had been on insulin for at least 6 months, with a baseline HbA1c of 7.0% (53 mmol/mol) or higher. Baseline characteristics and laboratory data from 495 patients were subjected to multiple regression analysis to identify factors influencing the change of HbA1c.
Results
Most patients (n = 809) received sitagliptin at a dose of 50 mg. In the 1,004 patients, HbA1c decreased by 0.74% (6 mmol/mol) and body weight increased by 0.1 kg after 6 months of combination therapy. Multiple regression analysis showed that a higher baseline HbA1c, older age, and lower body mass index influenced the change of HbA1c after 6 months. Hypoglycemic symptoms occurred in 7.4%, but none were severe.
Conclusions
These results emphasize the importance of a higher HbA1c at the commencement of sitagliptin therapy in patients on insulin. Glucose-dependent suppression of glucagon secretion by sitagliptin may be useful in patients with impaired insulin secretion. Sitagliptin can be used concomitantly with insulin irrespective of the insulin regimen, duration of insulin treatment, and concomitant medications.
doi:10.14740/jocmr2149w
PMCID: PMC4471747  PMID: 26124906
Type 2 diabetes; Sitagliptin; Insulin; Combination therapy; HbA1c; Multiple regression analysis; Body weight
16.  The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ 
eLife  null;4:e05615.
Adipocyte differentiation is a strictly controlled process regulated by a series of transcriptional activators. Adipogenic signals activate early adipogenic activators and facilitate the transient formation of early enhanceosomes at target genes. These enhancer regions are subsequently inherited by late enhanceosomes. PPARγ is one of the late adipogenic activators and is known as a master regulator of adipogenesis. However, the factors that regulate PPARγ expression remain to be elucidated. Here, we show that a novel ubiquitin E3 ligase, tripartite motif protein 23 (TRIM23), stabilizes PPARγ protein and mediates atypical polyubiquitin conjugation. TRIM23 knockdown caused a marked decrease in PPARγ protein abundance during preadipocyte differentiation, resulting in a severe defect in late adipogenic differentiation, whereas it did not affect the formation of early enhanceosomes. Our results suggest that TRIM23 plays a critical role in the switching from early to late adipogenic enhanceosomes by stabilizing PPARγ protein possibly via atypical polyubiquitin conjugation.
DOI: http://dx.doi.org/10.7554/eLife.05615.001
eLife digest
The world is facing a global epidemic of obesity, which also increases the risk for diabetes and heart disease. Obesity is caused when excess fat is stored in fat cells, and overweight individuals have larger fat cells compared to healthy weight people. Therefore understanding how fat cells are created in the body can provide new ways to combat obesity.
Fat cells, also known as adipocytes, arise from precursor cells via a process called adipogenesis. This requires the activity of proteins called transcription factors that bind to DNA and switch on the expression of genes. PPARγ is an important transcription factor that drives the expression of the genes that are needed to convert a precursor cell to a mature adipocyte.
For adipogenesis to proceed, cells have to maintain the appropriate levels of PPARγ. If the amount of PPARγ bound to DNA is too low, then it is unable to activate gene expression. However, the mechanisms by which cells maintain the correct levels of PPARγ activity remain poorly understood. Watanabe et al. analyzed this process in mouse cells and identified a protein called TRIM23 that is produced in precursor cells. Cells in which the levels of TRIM23 were artificially lowered failed to mature into fat cells; this suggests that this protein is necessary for adipogenesis. Furthermore, in the absence of TRIM23, the amount of PPARγ that occupied regions of DNA was also markedly reduced. A direct consequence of this was a decline in the expression of several genes that are required for the later steps in the adipogenesis process.
Watanabe et al. next analyzed the mechanism through which TRIM23 had an effect on the levels of PPARγ. It is known from previous work that TRIM23 belongs to a family of enzymes that attach a small molecular tag called ubiquitin onto other proteins. This ubiquitin tag typically marks these proteins for rapid destruction by a large molecular machine called the proteasome. Watanabe et al. found that TRIM23 also modified PPARγ with ubiquitin, but that it did so in an unusual manner that instead prevented the proteasome from recognizing PPARγ and destroying it. As such, TRIM23 stabilizes the levels of PPARγ in cells.
By providing new insights into how adipogenesis is regulated, these findings suggest that TRIM23 may be a potential therapeutic target in the treatment of diabetes and disorders related to obesity.
DOI: http://dx.doi.org/10.7554/eLife.05615.002
doi:10.7554/eLife.05615
PMCID: PMC4426667  PMID: 25905670
TRIM23; ubiquitin; PPARγ; adipocyte; transcription; human; mouse
17.  PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation 
Autophagy  2014;10(4):631-641.
Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin–proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.
doi:10.4161/auto.27785
PMCID: PMC4091150  PMID: 24451648
PARK2-mediated mitophagy; skeletal muscle atrophy; proteasome; NFE2L1; slow-twitch muscle; autophagy; mitochondria; knockout mouse
18.  Dimensionality of object representations in monkey inferotemporal cortex 
Neural computation  2014;26(10):2135-2162.
We have calculated the intrinsic dimensionality of visual object representations in anterior inferotemporal (AIT) cortex, based on responses of a large sample of cells stimulated with photographs of diverse objects. As dimensionality was dependent on data set size, we determined asymptotic dimensionality as both the number of neurons and number of stimulus image approached infinity. Our final dimensionality estimate was 93 (SD: ± 11), indicating that there is basis set of approximately a hundred independent features that characterize the dimensions of neural object space. We believe this is the first estimate of the dimensionality of neural visual representations based on single-cell neurophysiological data. The dimensionality of AIT object representations was much lower than the dimensionality of the stimuli. We suggest that there may be a gradual reduction in the dimensionality of object representations in neural populations going from retina to inferotemporal cortex, as receptive fields become increasingly complex.
doi:10.1162/NECO_a_00648
PMCID: PMC4191674  PMID: 25058707
19.  Time Context of Cue-Outcome Associations Represented by Neurons in Perirhinal Cortex 
The Journal of Neuroscience  2015;35(10):4350-4365.
The perirhinal cortex (PRh), which has extensive connections with diverse brain sites, may contribute to semantic memory by associating various types of information about objects. However, the extent of the types of associations in which PRh participates is unknown. In the present study, we let monkeys experience a consistent contingency between visual cues and different types of outcomes (water reward and sound-only acknowledgment) in a particular time context for many days and then recorded neuronal activities from PRh and area TE, which is the major source of visual inputs to PRh. We found that PRh cells represented the outcome type in their responses to the visual cues only in the time context in which the monkeys had experienced the cue-outcome contingency. In contrast, TE cells represented the outcome information whenever the cue appeared (i.e., independently from the related time context). These results showed that PRh cells represented not only the cue-outcome contingency but also the time context in which the monkeys had experienced the contingency. We conclude that PRh is not specific to the representation of sensory and associative properties of objects themselves but may represent broader information about objects, including the time context in which the objects are associated with particular outcomes.
doi:10.1523/JNEUROSCI.4730-14.2015
PMCID: PMC4355203  PMID: 25762680
area TE; inferotemporal cortex; macaque monkey; reward
20.  Unconventional PINK1 localization to the outer membrane of depolarized mitochondria drives Parkin recruitment 
Journal of Cell Science  2015;128(5):964-978.
ABSTRACT
Dysfunction of PTEN-induced putative kinase 1 (PINK1), a Ser/Thr kinase with an N-terminal mitochondrial-targeting sequence (MTS), causes familial recessive parkinsonism. Reduction of the mitochondrial membrane potential limits MTS-mediated matrix import and promotes PINK1 accumulation on the outer mitochondrial membrane (OMM) of depolarized mitochondria. PINK1 then undergoes autophosphorylation and phosphorylates ubiquitin and Parkin, a cytosolic ubiquitin ligase, for clearance of damaged mitochondria. The molecular basis for PINK1 localization on the OMM of depolarized mitochondria rather than release to the cytosol is poorly understood. Here, we disentangle the PINK1 localization mechanism using deletion mutants and a newly established constitutively active PINK1 mutant. Disruption of the MTS through N-terminal insertion of aspartic acid residues results in OMM localization of PINK1 in energized mitochondria. Unexpectedly, the MTS and putative transmembrane domain (TMD) are dispensable for OMM localization, whereas mitochondrial translocase Tom40 (also known as TOMM40) and an alternative mitochondrial localization signal that resides between the MTS and TMD are required. PINK1 utilizes a mitochondrial localization mechanism that is distinct from that of conventional MTS proteins and that presumably functions in conjunction with the Tom complex in OMM localization when the conventional N-terminal MTS is inhibited.
doi:10.1242/jcs.161000
PMCID: PMC4342580  PMID: 25609704
Mitochondria; Parkin; Parkinson's disease; PINK1
21.  Mitochondrial Complexes I and II Are More Susceptible to Autophagy Deficiency in Mouse β-Cells 
Endocrinology and Metabolism  2015;30(1):65-70.
Background
Damaged mitochondria are removed by autophagy. Therefore, impairment of autophagy induces the accumulation of damaged mitochondria and mitochondrial dysfunction in most mammalian cells. Here, we investigated mitochondrial function and the expression of mitochondrial complexes in autophagy-related 7 (Atg7)-deficient β-cells.
Methods
To evaluate the effect of autophagy deficiency on mitochondrial function in pancreatic β-cells, we isolated islets from Atg7F/F:RIP-Cre+ mice and wild-type littermates. Oxygen consumption rate and intracellular adenosine 5'-triphosphate (ATP) content were measured. The expression of mitochondrial complex genes in Atg7-deficient islets and in β-TC6 cells transfected with siAtg7 was measured by quantitative real-time polymerase chain reaction.
Results
Baseline oxygen consumption rate of Atg7-deficient islets was significantly lower than that of control islets (P<0.05). Intracellular ATP content of Atg7-deficient islets during glucose stimulation was also significantly lower than that of control islets (P<0.05). By Oxygraph-2k analysis, mitochondrial respiration in Atg7-deficient islets was significantly decreased overall, although state 3 respiration and responses to antimycin A were unaffected. The mRNA levels of mitochondrial complexes I, II, III, and V in Atg7-deficient islets were significantly lower than in control islets (P<0.05). Down-regulation of Atg7 in β-TC6 cells also reduced the expression of complexes I and II, with marginal significance (P<0.1).
Conclusion
Impairment of autophagy in pancreatic β-cells suppressed the expression of some mitochondrial respiratory complexes, and may contribute to mitochondrial dysfunction. Among the complexes, I and II seem to be most vulnerable to autophagy deficiency.
doi:10.3803/EnM.2015.30.1.65
PMCID: PMC4384673  PMID: 25325275
Autophagy; Insulin-secreting cells; Mitochondria; Mitochondrial complex
22.  An Effect of Molecular Motion on Carrier Formation in a Poly(3-hexylthiophene) Film 
Scientific Reports  2015;5:8436.
Free carriers, polarons (P), in conjugated polymers play a key role in the performance of optoelectronic devices. Here, we present solid evidence that P can be predominantly generated from polaron pairs (PP) in a poly(3-hexylthiophene) (P3HT) film under zero electric field. P formation from PP strongly depends on temperature. The temperature dependence of P starts to change around 300 K. P3HT exhibits a thermal molecular motion named the α1 relaxation process, in which the twisting motion of thiophene rings is released, in this temperature region. Thus, it can be claimed that the twisting motion of P3HT thiophene rings is one of the determining factors of the photodynamics of P in P3HT films. This finding holds true for poly(thiophene)s with different alkyl lengths and should be considered in the design and construction of highly-functionalized organic devices based on poly(thiophene)s.
doi:10.1038/srep08436
PMCID: PMC4327410  PMID: 25678470
23.  The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates 
Nature Communications  2015;6:6116.
Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.
Ubiquitin is a stable and soluble protein, but it is commonly found in inclusion bodies in neurodegenerative disorders and cancer. Here, Morimoto et al. report that increasing ubiquitin chain length leads to the formation of amyloid-like fibrils, which are degraded by an autophagy mechanism.
doi:10.1038/ncomms7116
PMCID: PMC4309437  PMID: 25600778
24.  Anatomical inputs to sulcal portions of areas 9m and 8Bm in the macaque monkey 
Neuronal activities recorded from the dorsal bank of the anterior cingulate sulcus have suggested that this cortical area is involved in control of search vs. repetition, goal-based action selection and encoding of prediction error regarding action value. In this study, to explore potential anatomical bases for these neuronal activities, we injected retrograde tracers (CTB-Alexa-488 and CTB-gold) into the dorsal bank of the anterior cingulate sulcus and examined the distribution of labeled cell bodies in macaque monkey brains. The Nissl staining showed that the cortex in the dorsal bank of the anterior cingulate sulcus has consistent layer 4 which means that the cortical region is a part of the granular prefrontal cortex. The injection site belonged to the sulcal portion of area 9m in two cases and the sulcal portion of area 8Bm in one case. In addition to the continuous distribution of labeled cells in the two areas (areas 9m and 8Bm) around the injection site, the labeled cells were densely distributed in the cingulate areas (areas 24, 32, and 23) in all the cases. The dense labeling of cells was also found in other prefrontal areas (areas 46, 10, 11, and 12) in the two cases with injection into the sulcal portion of area 9m, whereas the dense labeling of cells was found in pre-motor areas (F6 and F7) in the case with injection into the sulcal portion of area 8Bm. The dense labeling of cells in the prefrontal and premotor areas was more similar to those previously found after injections into dorsal parts of areas 9 and 8B. Subcortical distribution of labeled cells was found in the mediodorsal nucleus of thalamus, claustrum, and substantia nigra pars compacta in all the cases.
doi:10.3389/fnana.2015.00030
PMCID: PMC4357300  PMID: 25814938
retrograde tracer; labeled cell bodies; connections; area 9m; area 8Bm
25.  Neural Substrates of View-Invariant Object Recognition Developed without Experiencing Rotations of the Objects 
The Journal of Neuroscience  2014;34(45):15047-15059.
One fails to recognize an unfamiliar object across changes in viewing angle when it must be discriminated from similar distractor objects. View-invariant recognition gradually develops as the viewer repeatedly sees the objects in rotation. It is assumed that different views of each object are associated with one another while their successive appearance is experienced in rotation. However, natural experience of objects also contains ample opportunities to discriminate among objects at each of the multiple viewing angles. Our previous behavioral experiments showed that after experiencing a new set of object stimuli during a task that required only discrimination at each of four viewing angles at 30° intervals, monkeys could recognize the objects across changes in viewing angle up to 60°. By recording activities of neurons from the inferotemporal cortex after various types of preparatory experience, we here found a possible neural substrate for the monkeys' performance. For object sets that the monkeys had experienced during the task that required only discrimination at each of four viewing angles, many inferotemporal neurons showed object selectivity covering multiple views. The degree of view generalization found for these object sets was similar to that found for stimulus sets with which the monkeys had been trained to conduct view-invariant recognition. These results suggest that the experience of discriminating new objects in each of several viewing angles develops the partially view-generalized object selectivity distributed over many neurons in the inferotemporal cortex, which in turn bases the monkeys' emergent capability to discriminate the objects across changes in viewing angle.
doi:10.1523/JNEUROSCI.1898-14.2014
PMCID: PMC4220033  PMID: 25378169
monkey inferotemporal cortex; object; view invariance

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