Thrombospondin-1 protects against Aβ-induced mitochondrial fragmentation and dysfunction in hippocampal cells.

Alzheimer's disease (AD) is often characterized by the impairment of mitochondrial function caused by excessive mitochondrial fragmentation. Thrombospondin-1 (TSP-1), which is primarily secreted from astrocytes in the central nervous system (CNS), has been suggested to play a role in synaptogenesis, spine morphology, and synaptic density of neurons. In this study, we investigate the protective role of TSP-1 in the recovery of mitochondrial morphology and function in amyloid β (Aβ)-treated mouse hippocampal neuroblastoma cells (HT22). We observe that TSP-1 inhibits Aβ-induced mitochondrial fission by maintaining phosphorylated-Drp1 (p-Drp1) levels, which results in reduced Drp1 translocation to the mitochondria. By using gabapentin, a drug that antagonizes the interaction between TSP-1 and its neuronal receptor α2δ1, we observe that α2δ1 acts as one of the target receptors for TSP-1, and blocks the reduction of the p-Drp1 to Drp1 ratio, in the presence of Aβ. Taken together, TSP-1 appears to contribute to maintaining the balance in mitochondrial dynamics and mitochondrial functions, which is crucial for neuronal cell viability. These data suggest that TSP-1 may be a potential therapeutic target for AD

Inactivation of Medial Prefrontal Cortex Impairs Time Interval Discrimination in Rats

Several lines of evidence suggest the involvement of prefrontal cortex in time interval estimation. The underlying neural processes are poorly understood, however, in part because of the paucity of physiological studies. The goal of this study was to establish an interval timing task for physiological recordings in rats, and test the requirement of intact medial prefrontal cortex (mPFC) for performing the task. We established a temporal bisection procedure using six different time intervals ranging from 3018 to 4784 ms that needed to be discriminated as either long or short. Bilateral infusions of muscimol (GABAA receptor agonist) into the mPFC significantly impaired animal's performance in this task, even when the animals were required to discriminate between only the longest and shortest time intervals. These results show the requirement of intact mPFC in rats for time interval discrimination in the range of a few seconds

Genetic associations of in vivo pathology influence Alzheimers disease susceptibility

Introduction Although the heritability of sporadic Alzheimers disease (AD) is estimated to be 60–80%, addressing the genetic contribution to AD risk still remains elusive. More specifically, it remains unclear whether genetic variants are able to affect neurodegenerative brain features that can be addressed by in vivo imaging techniques. Methods Targeted sequencing analysis of the coding and UTR regions of 132 AD susceptibility genes was performed. Neuroimaging data using 11C-Pittsburgh Compound B positron emission tomography (PET), 18F-fluorodeoxyglucose PET, and MRI that are available from the KBASE (Korean Brain Aging Study for Early Diagnosis and Prediction of Alzheimers disease) cohort were acquired. A total of 557 participants consisted of 336 cognitively normal (CN) adults, 137 mild cognitive impairment (MCI), and 84 AD dementia (ADD) groups. Results We called 5391 high-quality single nucleotide variants (SNVs) on AD susceptibility genes and selected significant associations between variants and five in vivo AD pathologies: (1) amyloid β (Aβ) deposition, (2) AD-signature region cerebral glucose metabolism (AD-Cm), (3) posterior cingulate cortex (PCC) cerebral glucose metabolism (PCC-Cm), (4) AD-signature region cortical thickness (AD-Ct), and (5) hippocampal volume (Hv). The association analysis for common variants (allele frequency (AF) > 0.05) yielded several novel loci associated with Aβ deposition (PIWIL1-rs10848087), AD-Cm (NME8-rs2722372 and PSEN2-rs75733498), AD-Ct (PSEN1-rs7523) and, Hv (CASS4-rs3746625). Meanwhile, in a gene-based analysis for rare variants (AF < 0.05), cases carrying rare variants in LPL, FERMT2, NFAT5, DSG2, and ITPR1 displayed associations with the neuroimaging features. Exploratory voxel-based brain morphometry between the variant carriers and non-carriers was performed subsequently. Finally, we document a strong association of previously reported APOE variants with the in vivo AD pathologies and demonstrate that the variants exert a causal effect on AD susceptibility via neuroimaging features. Conclusions This study provides novel associations of genetic factors to Aβ accumulation and AD-related neurodegeneration to influence AD susceptibility.The study was supported by grants from the National Research Foundation of Korea (2014M3C7A1046049 and 2018M3C9A5064708 for Choi M and 2014M3C7A1046042 for Lee DY) and grants from the Ministry of Health and Welfare of Korea (HI18C0630 for Mook-Jung IH and Lee DY, and HI19C0149 for Lee DY)

Mitochondria-Specific Accumulation of Amyloid β Induces Mitochondrial Dysfunction Leading to Apoptotic Cell Death

Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ1–42 treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ1–42-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ1–42 in HT22 cells using Aβ1–42 with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ1–42-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ1–42 accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ1–42-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ1–42 accumulation, which mimics the apoptosis process in exogenous Aβ1–42-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ1–42 accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aβ-mediated signaling alterations

Investigating the mechanism of Aβ-induced mitochondrial dysfunctiion as a risk factor for Alzheimer's disease

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의과학과 의과학전공, 2016. 2. 묵인희.알츠하이머병의 가장 큰 특징은 세포 외부의 베타-아밀로이드 (Aβ) 플라크의 축적과 세포 내부의 과인산화된 타우 단백질의 비정상적인 접힘 현상을 들 수 있는데, 최근 이러한 현상학적인 세포 변화 뿐만 아니라 그에 의해 영향을 받는 생리적 기능의 이상이나 세포 소기관의 기능적 결함들이 직접적인 병인 기전으로써 많은 연구가 되고 있다. 미토콘드리아의 기능 이상과 autophagosome의 축적도 그들 중 하나이다. 하지만 아직까지 이에 대한 뚜렷한 메커니즘이 밝혀진 바가 없기에, 본 연구에서는 이를 구체적으로 밝히고자 하였다. 먼저, Crif1 (CR6 interacting factor 1) 이라는 단백질이 알츠하이머병 동물모델과 환자에서 감소되어 있음을 관찰하여 SY5Y 세포주에서 Aβ에 의해 감소된 Crif1의 양을 회복시켜 주었고 이때 미토콘드리아의 기능과 모양, 세포의 활성 등이 정상적으로 회복됨을 통해 Crif1이 미토콘드리아의 기능 유지를 위한 중요 단백질임인 동시에 알츠하이머병에서 주요 치료 표적으로 기능할 수 있음을 밝혀내었다. 또한, 뇌의 성상세포 (astrocyte) 에서 주로 분비되어 신경세포에 작용한다고 알려져 있는 Tsp-1 (Thrombospondin-1) 은 알츠하이머병 환자와 동물 모델에서 그 양이 감소되어 있는데, 본 연구에서 Tsp-1이 Aβ에 의한 미토콘드리아의 과분열 현상을 저해해 그에 따른 미토콘드리아의 기능 이상 및 세포 활성에 보호 효과를 낼 수 있음을 확인하였다. 이로써 Tsp1 또한 알츠하이머병 상황에서 미토콘드리아에의 보호 효과를 냄으로써 좋은 치료 표적이 될 수 있음을 확인하였다. 알츠하이머병은 제 3의 당뇨병이라고 불리어질 만큼 당뇨병과의 연관성이 깊은 것으로도 알려져 있다. 본 연구에서는 제 2형 당뇨병 치료제로 가장 흔히 쓰이는 metformin이 알츠하이머병 환자와 동물 모델에서 Aβ 생성을 증가시킴을 확인하고, HT22 세포주에서 metformin이 알츠하이머병의 주요 병인 기전 중의 하나인 autophagosome의 축적을 유도하여 Aβ 생성 증가에 기여할 수 있음을 확인하였다. 본 연구를 통해, 미토콘드리아의 구조적, 기능적 정상화가 알츠하이머병에서 중요한 치료 전략이 될 수 있음을 밝혀냄과 동시에, metformin이라는 제 2형 당뇨병 치료제가 그 병증을 가속화시킬 수 있는 가능성에 대해 제시할 수 있었다.Alzheimers disease (AD), a devastating form of dementia is the most common-age related neurological disorder. Extracellular Aβ accumulation and abnormal folding of intracellular hyper-phosphorylated tau proteins are two hall-marks of Alzheimers disease pathology. Recently, many studies have been focused on the maintenance of essential and principal physiological functions or the functional recovery of cellular organelles functions to increase the cell viability. Since mitochondrial dysfunction and accumnulation of autophagosomes are one of them, this study tried to reveal the mechanisms underlying between those risk factors and AD pathology progression. We found out that Aβ-induced disruption of mitochondrial morphology and function is mainly caused by Crif1 (CR6-interacting factor 1) loss. Furthermore, since Crif1 over-expression could recover Aβ-induced mitochondrial dysfunction, mitochondrial morphology disruption and decrease of cell viability in SH-SY5Y cells, Crif1 might be the good therapeutic target for AD maintaining mitochondrial functions. In addition, this study additionally confirmed that Tsp1 (Thrombospondin-1), which is released from astrocytes in CNS, appears to protect mitochondrial morphology and functions by inhibiting Aβ-induced calcium mediated calcineurin activation and following decrease of p-Drp1 level, which also can be a potential therapeutic strategy for AD. The association between type 2 diabetes and AD has been known to be quite strong. This study revealed the effect of metformin in Aβ production by showing that metformin accumulates autophagosomes where amyloidogenic APP processing is facilitated in HT22 cells. Based on these results, it is highly possible that maintaining mitochondrial function might be a good therapeutic approach in treating AD. Furthermore, this study also warns us for the effect of metformin that might contribute to AD pathology by enhancing Aβ generation.서론 1 Part 1. 알츠하이머병에서 관찰되는 Crif1 단백질의 감소로 초래되는 미토콘드리아의 기능 저해 연구 3 Part 2. Tsp-1의 A에 의한 미토콘드리아의 구조 망가짐 및 기능 이상에의 회복 기전 연구 4 Part 3. Metformin에 의한 A 생성 증가 메커니즘에 대한 연구 5 실험재료 및 방법 17 1. 실험에 이용된 실험동물 17 2. 제 2형 당뇨병 치료제인 metformin투여 방법 18 3. 세포주 및 세포배양 (Cell line and Cell culture) 18 4. 시약 (Reagents) 19 5. 플라스미드 DNA 및 siRNA 형질주입 (Transfection) 19 6. 미토콘드리아의 기능 분석 20 7. 미토콘드리아의 모양 분석 21 8. 웨스턴 블롯 (Western Blot) 22 9. 세포 활성 분석 실험 24 10. RNA 분리와 역전사 중합연쇄효소반응 (RT-PCR) 26 11. 정량적 실시간 중합연쇄효소반응 (qRT-PCR) 26 12. 전기이동 기동성 교대 실험 (EMSA) 27 13. 면역침강법 (Immunoprecipitation) 27 14. 면역조직염색 (Immunohistochemistry) 28 15. 세포 내 칼슘 측정 29 16. Luciferase reporter gene assay 30 17. BACE1 promoter activity assay 30 18. A ELISA assay 31 19. Trichloroacetic acid (TCA) precipitation 32 20. 전자현미경 관찰 32 21. Microsome enriched crude fraction 분리 33 22. In vitro peptide cleavage assay 34 23. A1-42 준비 (A1-42 preparation) 35 24. 통계처리 35 결과 36 Part 1. A에 의한 Crif1 단백질의 감소로 초래되는 미토콘드리아의 기능 저해 연구 36 1. 5XFAD 쥐와 알츠하이머병 환자의 병변 지역에서 보이는 Crif1 단백질의 저하 36 2. A에 의해 증가한 ROS가 Sp1의 전사 활성에 미치는 영향 38 3. 미토콘드리아 모양 유지에 있어서의 Crif1의 역할 40 4. Crif1의 감소가 미토콘드리아 기능에 미치는 영향 42 5. A에 의한 미토콘드리아 모양 망가짐과 기능 이상에 Crif1 과발현이 가지는 회복 효과 43 6. Crif1의 양이 A에 의한 세포 활성 감소에 미치는 영향 43 Part 2. Tsp1에 의한 알츠하이머병에서의 미토콘드리아 기능 이상 회복 연구 70 1. Tsp1이 A에 의한 미토콘드리아의 과도한 분열을 저해함을 확인 70 2. Tsp1이 A에 의한 미토콘드리아 기능 이상을 저해함을 확인 71 3. Tsp1이 A에 의한 신경 세포사를 저해함을 확인 71 4. Tsp1이 A에 의한 calcineurin 활성화와 p-Drp1 단백질의 감소를 저해함을 확인 72 Part 3. Metformin에 의한 A 생성 증가 메커니즘에 대한 연구 83 1. 5XFAD 쥐의 뇌에서 metformin의 A 플라크 형성 증가와 A42 생성 증가 효과 83 2. Metformin이 SH-SY5Y 세포에서 베타-, 감마-시크리테아제의 활성을 증가시켜 A를 증가시킴을 확인 84 3. Metformin이 SH-SY5Y세포에서 autophagosome을 축적시킴을 확인 85 4. Metformin에 의한 autophagosome 증가는 AMPK 신호전달 경로에 의존적임을 규명 86 고찰 96 Part 1. 알츠하이머병에서 관찰되는 Crif1 단백질의 감소로 초래되는 미토콘드리아의 기능 저해 연구 97 Part 2. Tsp1의 A에 의한 미토콘드리아의 구조 망가짐과 기능 이상에의 회복 기전 연구 102 Part 3. Metformin에 의한 A 생성 증가 메커니즘에 대한 연구 106 결론 113 참고문헌 116 Abstract 124Docto

Unmasking molecular profiles of bladder cancer

Precision medicine is designed to tailor treatments for individual patients by factoring in each person's specific biology and mechanism of disease. This paradigm shifted from a “one size fits all” approach to “personalized and precision care” requires multiple layers of molecular profiling of biomarkers for accurate diagnosis and prediction of treatment responses. Intensive studies are also being performed to understand the complex and dynamic molecular profiles of bladder cancer. These efforts involve looking bladder cancer mechanism at the multiple levels of the genome, epigenome, transcriptome, proteome, lipidome, metabolome etc. The aim of this short review is to outline the current technologies being used to investigate molecular profiles and discuss biomarker candidates that have been investigated as possible diagnostic and prognostic indicators of bladder cancer