Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells

In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. Previously, we have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis. Here, we report that phosphorylation of autophagy-associated protein p62 by AMP-activated protein kinase (AMPK) drives ACD and mitophagy in HCN cells. Pharmacological inhibition of AMPK or genetic ablation of the AMPK alpha 2 subunit by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing suppressed ACD, whereas AMPK activation promoted ACD in insulin-deprived HCN cells. We found that following insulin withdrawal AMPK phosphorylated p62 at a novel site, Ser-293/Ser-294 (in rat and human p62, respectively). Phosphorylated p62 translocated to mitochondria and induced mitophagy and ACD. Interestingly, p62 phosphorylation at Ser-293 was not required for staurosporine-induced apoptosis in HCN cells. To the best of our knowledge, this is the first report on the direct phosphorylation of p62 by AMPK. Our data suggest that AMPK-mediated p62 phosphorylation is an ACD-specific signaling event and provide novel mechanistic insight into the molecular mechanisms in ACD.1

Immunocytes as a Biocarrier to Delivery Therapeutic and Imaging Contrast Agents to Tumors

Radiotherapy for cancer treatment has been used for primary or adjuvant treatment in many types of cancer, and approximately half of all cancer patients are undergoing radiation. However, ionizing radiation exposure induces genetic alterations in cancer cells and results in recruitment of monocytes/macrophages by triggering signals released from these cells. Using this characteristic of monocytes/macrophages, we have attempted to develop a biocarrier loading radiosensitizing anticancer agents that can lead to enhance the therapeutic effect of radiation in cancer treatment. The aim of this study is to demonstrate the proof of this concept. THP-1 labeled with Qdot 800 or iron oxide (IO) effectively migrated into tumors of subcutaneous mouse model and increased recruitment after ionizing radiation. Functionalized liposomes carrying a radiosensitizing anticancer agent, doxorubicin, are successfully loaded in THP-1 (THP-1-LP-Dox) with reduced cytotoxicity, and THP-1-LP-Dox also was observed in tumors after intravenous administration. Here, we report that monocytes/macrophages as a biocarrier can be used as a selective tool for amplification of the therapeutic effects on radiotherapy for human cancer treatment

성체해마신경줄기세포의 자가포식현상에서 AMPK의 자가포식현상 조 절자로의 역할

AMP-activated protein kinase (AMPK) is a well-studied molecule for the control of cellular metabolism. In addition, AMPK can affect autophagy to sustain homeostasis. Adult hippocampal neural stem (HCN) cells undergo autophagic cell death (ACD) without apoptotic features following insulin withdrawal, therefore, insulin-deprived HCN cells are regarded as a genuine model for studying autophagy and cell death-related signaling mechanisms. In this study, we investigated the role of AMPK on autophagy regulation in HCN cells following insulin-withdrawal. Genetic and pharmacological activation of AMPK promoted ACD, while inhibition of AMPK yielded the opposite outcomes. These data suggest that AMPK can be a positive regulator of ACD in HCN cells following insulin withdrawal. ⓒ 2016 DGIST1. Introduction 1 -- 2. Materials and methods 5 -- 2.1 Cell culture 5 -- 2.2 Antibodies and reagents 5 -- 2.3 Cell death assay 5 -- 2.4 GFP-LC3 puncta assay 6 -- 2.5 Western blot 6 -- 2.6 Transfection 6 -- 2.7 Sh-RNA Lentiviral system 7 -- 2.8 Statistical analysis 7 -- 3. Results 8 -- 3.1 AMPK activator, A769662 increased cell death following insulin withdrawal. 8 -- 3.2 Ectopic expression of AMPK constructs modulated cell death. 8 -- 3.3 Silencing of AMPK reduced ACD in insulin-deprived HCN cells. 9 -- 3.4 Cell death mode induced by genetic and pharmacological activation of AMPK was ACD, not apoptosis. 9 -- 3.5 Suppression of autophagy related gene, Atg7 prevented ACD. 10 -- 4. Discussion 11 -- 5. Figure legends 13 -- 6. Figures 16 -- Reference 24 -- Abstract in Korean 26--장수 100세 시대에 이르며 퇴행성 뇌질환 환자가 증가하고 있으며, 뚜렷한 치료방법이 없어서 증가하는 환자에 대한 해결방안이 제대로 마련되지 않는 것이 사회적 문제로 대두되고 있다. 대표적 퇴행성 뇌질환인 알쯔하이머 치매나 파킨슨병과 같은 질병들은 신경세포들이 죽어서 수가 절대적으로 줄어드는 증상을 보인다. 자가포식현상은 포유류의 몸 속에서 대사산물로 발생한 몸에 필요치 않는 물질들을 없애기 위한 방법으로 세포 내 자기항상성에 기여를 하는 아주 중요한 과정이다. 그러나 어떤 이유로 인해 체내 자가포식현상이 정상적으로 일어나지 않으면, 많은 질병 특히나 퇴행성 뇌질환을 야기한다는 연구들이 많이 진행되었다. 하지만 중요도에 비해 정확한 자가포식현상, 궁극적으로 세포사멸 기전에 대한 이해가 부족해 더 많은 연구가 이루어져야 한다고 보고 있다. 본 논문은 성체해마신경줄기세포의 세포사 기전 중에서도 세포 내 항상성을 유지하는 역할을 하는, 또한 비교적 이른 시간에 반응하는 AMPK라는 단백질의 자가포식현상에 미치는 영향에 대해 다루었다. 성체해마신경줄기세포에서 인슐린 결핍 시 자가포식현상만이 일어나므로, 성체해마신경 줄기세포를 이용하였다. 이 세포에서 AMPK가 인슐린 결핍상황에서 일어나는 자가포식현상을 증가시키는 것을 확인하였다. 단순히 현상뿐 아니라 유전적인 방법과 약물처리 등 여러 방법을 이용하여, 증가된 자가포식현상이 AMPK에 의해 조절된 것임을 증명하였다. 더 나아가 이 연구는 충분하지 못한 자가포식현상으로 인해 일어나는 퇴행성 뇌질환 또는 불필요하게 과활성화 된 뇌 세포에서의 자가포식현상을 조절하기 위해, AMPK 촉진제 또는 억제제를 사용한다면 치료제로도 충분히 사용할 수 있을 것이라는 가능성을 보여주고 있다. ⓒ 2016 DGISTMasterdCollectio

Distinct Signaling Pathways for Autophagy-Driven Cell Death and Survival in Adult Hippocampal Neural Stem Cells

autophagy; autophagic cell death; ERK; JNK; GSK-3β; adult hippocampal neural stem cellsAutophagy is a conserved cellular catabolic process responsible for degrading and recycling cellular materials and plays beneficial roles in maintaining cellular and organismal homeostasis by preventing the accumulation of toxic protein aggregates, removing damaged organelles, and providing bioenergetic substrates essential for survival. However, autophagy can also induce cell death depending on the cellular context, and the signaling pathways differentially regulating these opposite outcomes remain largely unknown. Our previous study demonstrated that insulin withdrawal (IW) or corticosterone (CORT) induces autophagic cell death in adult hippocampal neural stem cells. In contrast, metabolic stresses caused by 2-deoxy-D-glucose (2DG) and glucose-low (GL) induce cytoprotective autophagy without cell death. By comparing these conditions, we revealed that distinct signaling pathways regulate cell death-inducing autophagy and survival-promoting autophagy, with ERK and JNK involved in the latter and GSK-3β in the former. Furthermore, our observations revealed mitochondrial dysfunction during death-inducing autophagy, whereas protective autophagy was associated with lipophagy. These findings suggest that autophagy regulates cell death and survival in HCN cells via distinct signaling pathways.|자가포식은 세포 소재를 분해하고 재활용하는 세포 대사과정으로, 독성 단백집합체의 축적 방지, 손상된 세포기관 제거, 생존에 필수적인 생체 에너지 기질 제공 등의 이점이 있다. 그러나 어떠한 이유에서, 조건에 따라 자가포식현상은 세포 사멸 또는 보호적인 역할을 다르게 하는 것이 관찰되었다. 반대되는 현상의 결과를 각각 조절하는 신호전달 경로는 아직 잘 알려져 있지 않았다. 기존에 우리 연구실에서는 인슐린부족(IW) 또는 코티코스테로이드 주입(CORT)에 의해 성인 해마 신경줄기세포가 자가포식현상이 증가하며 죽는 것을 발견했다. 이 연구에서는 앞선 연구와는 달리 2-Deoxy-D-Glucose (2DG)와 적은 포도당(GL) 조건에서는 자가포식현상에도 세포가 죽지 않았고, 오히려 자가포식현상이 세포 보호에 기여하는 것을 발견하였다. 우리는 IW 및 CORT 조건과 2DG 및 GL처리를 비교하여, 보호적인-자가포식을 유도하는 신호전달체계는 ERK 및 JNK가 관여하는데 반해, 세포사멸-자가포식을 조절하는 신호전달체계는 GSK-3β가 관여한다는 것을 밝혀냈다. 또한, 죽는 자가포식현상에서는 미토콘드리아 기능 이상이 나타났으며, 보호적인 자가포식현상은 지방 포식(Lipophagy)이 관련되어 있는 것을 확인하였다. 이러한 결과는 성인 해마 신경줄기세포에서 세포사멸과 생존 촉진 자가포식현상이 서로 다른 신호전달 경로를 통해 조절될 수 있다는 것을 시사한다. 본 연구는 자가포식 연구분야에서 논란이 많은 자가포식현상의 역할, ‘죽음을 야기하는가 혹은 보호적인 영향을 미치는가’에 대한 새로운 단서를 제공한다. 지금껏 우리가 아는 연구범위에서는 한 세포내에서 두가지 다른 자가포식현상의 발생을 처음 보고하는 연구이며 본 연구를 통해 다른 신호전달체계에 의한 조절이 자가포식의 세포사멸을 혹은 생존을 유도하는데 관련이 있고, 조절될 수 있다는 가능성을 제시한다.Ⅰ. Introduction 1 1. Cell death mechanism 1 1.1 Apoptosis 3 1.2. Necrosis 3 1.3. Autophagy 4 2. Autophagy in adult hippocampal neural stem cells 9 2.1 Death-inducing autophagy 12 2.1.1 Mitochondria – ATP, Mitochondrial membrane potential, reactive oxygen stress 12 2.1.2 Glycolysis / Gluconeogenesis 14 2.1.3 GSK-3β 17 2.2 Protective autophagy 17 2.2.1 Lipid droplet, lipophagy 17 2.2.2 MAPK 20 3. Aim/hypothesis/significance of this study 21 Ⅱ. Materials and Methods 23 1. Materials 23 2. Cell cultures 23 3. Cell death assay 23 4. Generation of stable cell lines 24 5. Transfection 24 6. Confocal imaging 25 7. Western blotting 25 8. MitoSOX staining using fluorescence-activated cell sorting (FACS) analysis 25 9. ATP measurement assay 26 10. Quantitative real-time PCR 26 11. Statistical analysis 26 Ⅲ. Results 28 Result 1. Autophagy is induced by four cellular stress conditions in HCN cells. 28 Result 2. 2DG and GL did not affect cell viability, whereas IW and CORT triggered autophagic cell death. 30 Result 3. 2DG-induced autophagy acted protectively and prevented IW- or CORT-induced cell death. 34 Result 4. MAPK inhibition prevents 2DG-induced autophagy but has no effect on IW- or CORT-induced autophagy. 38 Result 5. The blockade of ERK and JNK impedes the protective effects of 2DG. 38 Result 6. The inhibition of GSK-3β blocks IW- or CORT-induced ACD but not 2DG-induced autophagy. 44 Result 7. IW and CORT induce ATP depletion and mitochondrial ROS accumulation in HCN cells, unlike 2DG or GL treatment. 47 Result 8. Gluconeogenesis genes, but not glycolysis genes were up-regulated after ACD. 51 Result 9. Lipophagy is associated with MAPK-driven survival autophagy. 54 Ⅳ. Discussion 59 References 62 Abstract in Korean 82DoctordCollectio

Distinct Signaling Pathways for Autophagy-Driven Cell Death and Survival in Adult Hippocampal Neural Stem Cells

Autophagy is a cellular catabolic process that degrades and recycles cellular materials. Autophagy is considered to be beneficial to the cell and organism by preventing the accumulation of toxic protein aggregates, removing damaged organelles, and providing bioenergetic substrates that are necessary for survival. However, autophagy can also cause cell death depending on cellular contexts. Yet, little is known about the signaling pathways that differentially regulate the opposite outcomes of autophagy. We have previously reported that insulin withdrawal (IW) or corticosterone (CORT) induces autophagic cell death (ACD) in adult hippocampal neural stem (HCN) cells. On the other hand, metabolic stresses caused by 2-deoxy-D-glucose (2DG) and glucose-low (GL) induce autophagy without death in HCN cells. Rather, we found that 2DG-induced autophagy was cytoprotective. By comparing IW and CORT conditions with 2DG treatment, we revealed that ERK and JNK are involved with 2DG-induced protective autophagy, whereas GSK-3β regulates death-inducing autophagy. These data suggest that cell death and survival-promoting autophagy undergo differential regulation with distinct signaling pathways in HCN cells. © 2023 by the authors.TRU

Surface Electromyography-Driven Parameters for Representing Muscle Mass and Strength

The need for developing a simple and effective assessment tool for muscle mass has been increasing in a rapidly aging society. This study aimed to evaluate the feasibility of the surface electromyography (sEMG) parameters for estimating muscle mass. Overall, 212 healthy volunteers participated in this study. Maximal voluntary contraction (MVC) strength and root mean square (RMS) values of motor unit potentials from surface electrodes on each muscle (biceps brachii, triceps brachii, biceps femoris, rectus femoris) during isometric exercises of elbow flexion (EF), elbow extension (EE), knee flexion (KF), knee extension (KE) were acquired. New variables (MeanRMS, MaxRMS, and RatioRMS) were calculated from RMS values according to each exercise. Bioimpedance analysis (BIA) was performed to determine the segmental lean mass (SLM), segmental fat mass (SFM), and appendicular skeletal muscle mass (ASM). Muscle thicknesses were measured using ultrasonography (US). sEMG parameters showed positive correlations with MVC strength, SLM, ASM, and muscle thickness measured by US, but showed negative correlations with SFM. An equation was developed for ASM: ASM = −26.04 + 20.345 × Height + 0.178 × weight − 2.065 × (1, if female; 0, if male) + 0.327 × RatioRMS(KF) + 0.965 × MeanRMS(EE) (SEE = 1.167, adjusted R2 = 0.934). sEMG parameters in controlled conditions may represent overall muscle strength and muscle mass in healthy individuals

p Long-Term Human Histologic Evaluation of Sinus Bone Augmentation and Simultaneous Implant Placement

This study aimed to histologically and histomorphometrically evaluate osseointegration following simultaneous implant placement and maxillary sinus augmentation. Three retrospective human cases are described in which implants were placed at the maxillary sinus site augmented with deproteinized bovine bone mineral (DBBM) and later retrieved due to implant fracture after 5 to 8 years of occlusal loading. The removed implants with bone were processed for histologic evaluation, and bone-to-implant contact (BIC), bone area (BA), and mirror-image bone area (MIBA) were measured. Mature lamella bone was mainly observed, and some unabsorbed grafted bone particles remained in all cases. The measured values of BIC, BA, and MIBA in the three consecutive threads with the highest values were 86.0% to 91.2%, 65.8% to 91.9%, and 73.0% to 90.4%, respectively, and there were no signs of inflammation. Within the limits of this study, these cases demonstrate successful bone formation after maxillary sinus bone augmentation with DBBM and simultaneous implant placement. Int J Periodontics Restorative Dent 2022;42:93-100. doi:N