신경세포에서 프로피온산이 리소솜 손상에 미치는 영향과 L-세린을 통한 카텝신 D의 기능 회복에 대한 연구

L-serine, lysosome, lipid droplet, propionic acid (PPA), neuronL-serine is a non-essential amino acid endogenously produced by astrocytes and is abundant in human diets. Beneficial roles of the metabolic products from L-serine in various conditions in the brain including neuronal development have been reported. Through several preclinical studies, L-serine treatment was also shown to offer beneficial therapeutic effects for brain damage such as ischemic stroke, amyotrophic lateral sclerosis, and Parkinson’s disease. Despite evidence for the value of L-serine in the clinic, however, its beneficial effects on the propionic acid (PPA)-induced neuronal toxicity and underlying mechanisms of L-serine-mediated neuroprotection are unknown. In this study, I observed that PPA-induced acidic stress induces abnormal lipid accumulation and functional defects in lysosomes of hippocampal neurons. L-serine treatment was able to rescue the structure and function of lysosomes in PPA-treated hippocampal neuronal cells. I further identified that L-serine suppressed the formation of lipid droplets and abnormal lipid membrane accumulations inside the lysosomes in PPA-treated hippocampal neuronal cells. Taken together, these findings indicate that L-serine can be utilized as a neuroprotective agent for the functionality of lysosomes through restoration of cathepsin D in disease conditions.L-세린 (L-serine)은 성상교세포에 의해 내부적으로 생성되는 비 필수적인 아미노산이며, 일반적으로 우리가 섭취하는 음식물에도 많이 함유되어 있다. 신경 발달을 비롯하여L-세린이 뇌세포에 미치는 유익한 역할이 많은 연구에서 보고되고 있다. 여러 전임상 연구를 통해 L-세린을 이용한 치료는 허혈성 뇌졸중, 근위축성 축삭 경화증 및 파킨슨병과 같은 뇌 손상에 유익한 치료 효과를 제공하는 것으로 나타났다. 그러나 임상적 측면에서의 L-세린에 대한 다양한 효과에도 불구하고 프로피온산에 의한 신경 독성과 L-세린에 의한 손상된 신경을 회복하고, 보호하는 메커니즘에 대한 연구는 알려져 있지 않은 실정이다. 프로피온산은 장내 세균이 음식물을 발효시켜 만들어지는 분해 산물로써 탄소수가 6개 미만인 지방산이다. 정상인에 비해 자폐증 환자의 분변에서 프로피온산의 농도가 높은 것으로 보고되고 있으며, 높은 프로피온산 농도로 인해 프로피온산증을 동반하는 경우가 많다. 프로피온산을 쥐에게 투여한 경우 자폐증과 유사한 신경 및 행동학적 경향을 보이는 연구가 많이 보고되었으나, 신경세포에 미치는 영향에 대한 연구는 부족한 실정이다. 본 연구에서는 프로피온산으로 인한 산성 스트레스가 해마 뉴런에 미치는 영향 중 비정상적인 지질 축적과 리소솜의 기능적 결함을 유발한다는 것을 확인하였다. 광학 현미경의 분해능 한계를 극복하고자 광학현미경과 전자현미경을 연계한 분석법을 활용하여 신경세포내의 소기관 미세구조 변화를 고해상도 이미지를 통해 분석하였다. 컨포컬 현미경을 이용하여 신경세포에 존재하는 리소솜과 지방 방울의 위치를 형광 염색으로 확인하였고, 특정된 위치를 전자현미경의 고해상도 이미지를 얻어 구조적 변화를 분석하였다. 특히 연속절편법을 이용한 고해상도 전자현미경 이미지를 3차원 이미지로 재구성하여 2차원적 평면 이미지 분석의 한계를 극복하였다. L-세린을 해마 신경 세포에 처리하였을 때, 프로피온산에 의해 손상된 리소솜의 구조와 기능이 회복됨을 확인할 수 있었다. 또한 L-세린은 프로피온산에 의해 형성된 리소솜 내부의 지질 방울과 비정상적인 지질막 축적을 억제시킨다는 것을 확인하였다. 위의 결과들을 종합해보면, L-세린은 프로피온산에 의한 산성 스트레스 상태에서 손상된 리소솜의 기능을 회복시키는 역할을 하는데, 카텝신 D (Cathepsin D)라고 알려진 단백질 분해 효소의 활성화를 증가시켜 리소솜의 분해 기능을 회복시킨다는 것을 확인하였고, 이를 통해 L-세린은 신경 보호제로 사용될 수 있음을 유추해 볼 수 있다.I. Introduction 1 1.1 Introduction 1 1.2 Autism spectrum disorder associated with short chain fatty acids 3 1.3 Correlative light and electron microscopy (CLEM) 4 II. Materials and methods 6 2.1 Materials 6 2.1.1 Pharmacological treatment 6 2.2 Methods 6 2.2.1 Cell culture 6 2.2.2 Cell viability assays 7 2.2.3 Trypan blue staining 7 2.2.4 Lysosomal activity assays 8 2.2.5 Immunocytochemistry 9 2.2.6 Confocal microscopy analysis 9 2.2.7 Western blotting analysis 10 2.2.8 Transmission electron microscopy (TEM) 11 2.2.9 Three-dimensional reconstruction of serial TEM images 12 2.2.10 Statistical analysis 12 III. Results 13 3.1 Toxicological effects of propionic acid and l-serine on HT22 cells 13 3.2 PPA decreased lysosomal activity in PPA-treated HT22 cells, and l-serine attenuated the dysfunction 15 3.3 PPA increased lipid droplets in HT22 cells, and l-serine attenuated the lipid accumulation 20 3.4 Three-dimensional volume reconstruction in serial TEM showed effects of l-serine in decreasing lipid droplets in PPA-treated HT22 cells 23 3.5 L-serine decreased the colocalization of lipi-red and lysotracker in PPA-treated hippocampal neurons 27 3.6 Attenuation of lysosomal dysfunction by l-serine in PPA-treated hippocampal neurons 32 3.7 CLEM and serial TEM showed increase in lysosomal lipid membrane accumulation by PPA in hippocampal neurons 37 IV. Discussion and conclusion 42 V. References 46DoctordCollectio

Restoration of Cathepsin D Level via L-Serine Attenuates PPA-Induced Lysosomal Dysfunction in Neuronal Cells

L-serine is a non-essential amino acid endogenously produced by astrocytes and is abundant in human diets. Beneficial roles of the metabolic products from L-serine in various conditions in the brain including neuronal development have been reported. Through several preclinical studies, L-serine treatment was also shown to offer beneficial therapeutic effects for brain damage such as ischemic stroke, amyotrophic lateral sclerosis, and Parkinson’s disease. Despite evidence for the value of L-serine in the clinic, however, its beneficial effects on the propionic acid (PPA)-induced neuronal toxicity and underlying mechanisms of L-serine-mediated neuroprotection are unknown. In this study, we observed that PPA-induced acidic stress induces abnormal lipid accumulation and functional defects in lysosomes of hippocampal neurons. L-serine treatment was able to rescue the structure and function of lysosomes in PPA-treated hippocampal neuronal cells. We further identified that L-serine suppressed the formation of lipid droplets and abnormal lipid membrane accumulations inside the lysosomes in PPA-treated hippocampal neuronal cells. Taken together, these findings indicate that L-serine can be utilized as a neuroprotective agent for the functionality of lysosomes through restoration of cathepsin D in disease conditions

Phospholipid-based nanodrill technology for enhanced intracellular delivery of nano-sized cargos

Nanosized drug delivery systems typically enter the cell via endocytosis. However, a significant amount of the endocytosed cargo cannot effectively escape from the endosome, resulting in drug degradation. Therefore, there are several ongoing efforts to develop transmembrane delivery systems that could circumvent endocytosis. In this study, phospholipid nanotube nanodrills (LDs) were formed onto the surface of a human serum albumin nanoparticle via self-assembling phospholipids. The nanodrill technology enhanced the intracellular uptake efficiency of nanoparticles via energy-independent direct cell membrane permeation. The length of the nanodrills according to the DSPE-PEG to DSPC ratio was investigated both experimentally and theoretically. Our findings demonstrated that longer nanodrills were formed on the surface of the nanoparticles as the ratio of DSPC (i.e., a strongly hydrophobic lipid) in the two phospholipids increases. Moreover, the intracellular uptake efficiency increased as the length of phospholipid nanodrills increased. In addition to enhancing intracellular delivery, the phospholipid nanodrills could penetrate the extracellular matrix and enable the introduction of nanoparticles, thus highlighting the promising tissue penetration capacity of phospholipid nanodrill technology. The improved cell permeability of LD technology was demonstrated by effectively inhibiting specific genes via siRNA-based therapeutic delivery. Moreover, this approach enhanced the efficacy of chemotherapeutics against chemo-resistant cancer cells. Therefore, LD technology could be used to deliver genetic materials and chemical-based therapeutics both in vitro and in vivo. © 2023 The AuthorsTRU

Convolutional Neural Network Classifies Pathological Voice Change in Laryngeal Cancer with High Accuracy

Voice changes may be the earliest signs in laryngeal cancer. We investigated whether automated voice signal analysis can be used to distinguish patients with laryngeal cancer from healthy subjects. We extracted features using the software package for speech analysis in phonetics (PRAAT) and calculated the Mel-frequency cepstral coefficients (MFCCs) from voice samples of a vowel sound of /a:/. The proposed method was tested with six algorithms: support vector machine (SVM), extreme gradient boosting (XGBoost), light gradient boosted machine (LGBM), artificial neural network (ANN), one-dimensional convolutional neural network (1D-CNN) and two-dimensional convolutional neural network (2D-CNN). Their performances were evaluated in terms of accuracy, sensitivity, and specificity. The result was compared with human performance. A total of four volunteers, two of whom were trained laryngologists, rated the same files. The 1D-CNN showed the highest accuracy of 85% and sensitivity and sensitivity and specificity levels of 78% and 93%. The two laryngologists achieved accuracy of 69.9% but sensitivity levels of 44%. Automated analysis of voice signals could differentiate subjects with laryngeal cancer from those of healthy subjects with higher diagnostic properties than those performed by the four volunteers.11Ysciescopu

Controlling side-chain density of electron donating polymers for improving their packing structure and photovoltaic performance

A simple and efficient approach of controlling the side-chain density in the electron donating polymers has been demonstrated to tune their 3-D packing structure and HOMO level, which increases the hole mobility and V-oc values, thus improving the solar cell performance

Mosaic RBD nanoparticles induce intergenus cross-reactive antibodies and protect against SARS-CoV-2 challenge

Recurrent spillovers of α- and β-coronaviruses (CoV) such as severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome-CoV, SARS-CoV-2, and possibly human CoV have caused serious morbidity and mortality worldwide. In this study, six receptor-binding domains (RBDs) derived from α- and β-CoV that are considered to have originated from animals and cross-infected humans were linked to a heterotrimeric scaffold, proliferating cell nuclear antigen (PCNA) subunits, PCNA1, PCNA2, and PCNA3. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens, like jewels in a crown. Prime-boost immunizations with 6RBD-np in mice induced significantly high Ab titers against RBD antigens derived from α- and β-CoV and increased interferon (IFN-γ) production, with full protection against the SARS-CoV-2 wild type and Delta challenges. The mosaic 6RBD-np has the potential to induce intergenus cross-reactivity and to be developed as a pan-CoV vaccine against future CoV spillovers.</jats:p