Induction and attenuation of neuronal apoptosis by proteasome inhibitors in murine cortical cell cultures

Suh, Jaehong,Lee, Young Ae,Gwag, Byoung Joo Blackwell Science Ltd 2005 Journal of Neurochemistry Vol.95 No.3

<P>Abstract</P><P>Evidence has accumulated showing that pharmacological inhibition of proteasome activity can both induce and prevent neuronal apoptosis. We tested the hypothesis that these paradoxical effects of proteasome inhibitors depend on the degree of reduced proteasome activity and investigated underlying mechanisms. Murine cortical cell cultures exposed to 0.1 µ<SMALL>M</SMALL> MG132 underwent widespread neuronal apoptosis and showed partial inhibition of proteasome activity down to 30–50%. Interestingly, administration of 1–10 µ<SMALL>M</SMALL> MG132 almost completely blocked proteasome activity but resulted in reduced neuronal apoptosis. Similar results were produced in cortical cultures exposed to other proteasome inhibitors, proteasome inhibitor I and lactacystin. Administration of 0.1 µ<SMALL>M</SMALL> MG132 led to activation of a mitochondria-dependent apoptotic signaling cascade involving cytochrome <I>c</I>, caspase-9, caspase-3 and degradation of tau protein; such activation was markedly reduced with 10 µ<SMALL>M</SMALL> MG132. High doses of MG132 prevented the degradation of inhibitor of apoptosis proteins (IAPs) cIAP and X chromosome-linked IAP, suggesting that complete blockade of proteasome activity interferes with progression of apoptosis. In support of this, addition of high doses of proteasome inhibitors attenuated apoptosis of cortical neurons deprived of serum. Taken together, the present results indicate that inhibition of proteasome activity can induce or prevent neuronal cell apoptosis through regulation of mitochondria-mediated apoptotic pathways and IAPs.</P>

Role of p38 MAPK in the Regulation of Apoptosis Signaling Induced by TNF-α in Differentiated PC12 Cells

Park, Jung-Gyu,Yuk, Youn-Jung,Rhim, Hye-When,Yi, Seh-Yoon,Yoo, Young-Sook 생화학분자생물학회 2002 Journal of biochemistry and molecular biology Vol.35 No.3

TNF-$\alpha$ elicits various responses including apoptosis, proliferation, and differentiation according to cell type. In neuronal PC12 cells, TNF-$\alpha$ induces moderate apoptosis while lipopolysarccaharide or trophic factor deprivation can potentiate apoptosis that is induced by TNF-$\alpha$. TNF-$\alpha$ initiates various signal transduction pathways leading to the activation of the caspase family, NF-${\kappa}B$, Jun N-terminal kinase, and p38 MAPK via the death domain that contains the TNF-$\alpha$ receptor. Inhibition of translation using cycloheximide greatly enhanced the apoptotic effect of TNF-$\alpha$. This implies that the induction of anti-apoptotic genes for survival by TNF-$\alpha$ may be able to protect PC12 cells from apoptosis. Accordingly, Bcl-2, an anti-apoptotic genes for survival by TNF-$\alpha$ may be able to protect PC12 cells from apoptosis. Accordingly, Bcl-2, an anti-apoptotic Bcl-2 family member, was highly expressed in response to TNF-$\alpha$. In this study, we examined the anti-apoptotic role of p38 MAPK that is activated by TNF-$\alpha$ in neuronal PC12 cells. The phosphorylation of p38 MAPK in response to TNF-$\alpha$ slowly increased and lasted several hours in the PC12 cell and DRG neuron. This specific inhibitor of p38 MAPK, SB202190, significantly enhanced the apoptosis that was induced by TNF-$\alpha$ in PC12 cells. This indicates that the activation of p38 MAPK could protect PC12 cells from apoptosis since there is no known role of p38 MAPK in resoonse to TNF-$\alpha$ in neuron. This discovery could be evidence for the neuroprotective role of the p38 MAPK.

대뇌 기저핵 신경세포 일차배양에서 망간에 의해 유도된 Appoptosis : 형태학적인 소견

신동훈,김상표,정용욱,배재훈,송대규,백원기 大韓産業醫學會 2000 대한직업환경의학회지 Vol.12 No.1

목적 : 본 실험은 대뇌기저핵의 신경세포를 배양하여 망간(MnCl₂)을 투여한 후 망간독성에 의한 신경세포의 apoptosis를 형태학적인 소견으로 관찰하였다. 방법 : 배양된 신경세포에 0.01에서 10μM Mnc12를 48시간동안 처리한 후 TUNEL (TdT-mediated dUTP Nick End Labelling)법 및 투과전자현미경학적으로 관찰하였다. 결과 : TUNEL방법을 이용하여 관찰한 결과 TUNEL반응에 갈색으로 양성반응을 나타내는apoptotic 세포의 수가 대조군에 비해 MnCl₂를 투여한 군에서 유의하게 높게 나타났으며(P〈0.05), 투과전자현미경학적 소견상 대조군의 신경세포들은 핵인(nucleolus)이 두드러지게 특징적으로 보이면서 핵막과 세포질내 소기관들이 잘 보존되어 있으며, 세포질내망(ER)과 사립체(mitochondria)를 특히 많이 가지고 있었다. Mnc12를 48시간 동안 처리한 군에서 이질염색질(heterochromatin)이 핵막으로 이동하면서 응집되어 있었으며, 핵내 불규칙한 형태의 염색질이 나타나 분절이 진행되는 소견을 보였고, Apoptosis의 가장 특징적인 초기 소견인 막으로 둘러싸인 반달모양의 핵내염색질의 분절편(fragment-ed chromatin)과 주위의 상대적으로 정상적인 소기관으로 구성된 apoptotic body를 관찰할 수 있었다. 결론 : 신경세포에서 망간에 의해 apoptosis가 유도됨을 형태학적인 방법으로 확인할 수 있었으며 망간에 의한 세포사망양상에 apoptosis가 하나의 기전이 될 수 있을 것이다. Objectives : Manganese is cytotoxic to the central nervous system including basal gan-glia. Its toxic mechanism is related to oxidative stress, mediated by toxic free radicals but is specultives. In the present study , we have investigated to manifest apoptosis in man-ganese-induced cytotoxicity in primary neuronal cell culture of rat basal ganglia. Methods : To detect apoptotic neuronal cells were stained by the terminal deoxynu-cleotide (TdT) -mediated dUTP nick end-labelling (TUNEL) method and apoptotic changesin nuclei of neurons were observed by electron microscopy. Results : We showed that TUNEL immunostain showed brownish signal in the nuclei of apoptotic cells and the proportions of apoptotic cells in Manganese treatment groups were more higher than controls. On transmission electron microscopy, there were chromatine condensation with margination toward nuclear membrane and condensation of cyto-plasm in the treated with luM MnCl²for 48 hours in a basal ganglia neurons. Apoptotic bodies were found and consisted of semilunar-like condensed nuclei with relatively intact cytoplasmic organelles. Conclusions : Apoptosis appears to be one mechanism in the manganese-induced net-ronal cell death. Manganese intoxication is a convenient model for apoptosis study.

Fucoidan ameliorates amyloid-β 42 oligomer-induced neuronal apoptosis by activating the PI3K/Akt signaling pathway and MAPK cascades in human neuroblastoma SH-SY5Y cells

Kang Chae-Won,박미의,Lee Hae-Jeung 대한독성 유전단백체 학회 2024 Molecular & cellular toxicology Vol.20 No.3

Background Fucoidan, a polysaccharide derived from brown seaweed, has multiple biological properties, including antioxidant, antitumor, anticoagulant, and anti-inflammatory effects. However, there are few reports on their neuroprotective effects. Objective This study is about the neuroprotective effects of fucoidan against Aβ42-induced neuronal apoptosis in human neuroblastoma SH-SY5Y cells. Methods The protective effects of fucoidan against cell death by Aβ42 were measured with FDA/PI and Hoechst 33342 staining and immunoblotting about apoptosis-related proteins. Results In this study, fucoidan activated autophagy by regulating the expression of autophagy markers, significantly upregulating the Bcl-2/Bax ratio, and downregulating cleaved caspase-3/caspase-3. Moreover, fucoidan improved Aβ42-induced neuronal apoptosis by activating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway and mitogen-activated protein kinase cascade in SH-SY5Y cells. Additionally, Hoechst 33342 staining showed that fucoidan diminished the level of the apoptotic nuclei in Aβ42-induced SH-SY5Ycells. Conclusion This study proposes fucoidan as a potential therapeutic candidate for AD treatment. Background Fucoidan, a polysaccharide derived from brown seaweed, has multiple biological properties, including antioxidant, antitumor, anticoagulant, and anti-inflammatory effects. However, there are few reports on their neuroprotective effects. Objective This study is about the neuroprotective effects of fucoidan against Aβ42-induced neuronal apoptosis in human neuroblastoma SH-SY5Y cells. Methods The protective effects of fucoidan against cell death by Aβ42 were measured with FDA/PI and Hoechst 33342 staining and immunoblotting about apoptosis-related proteins. Results In this study, fucoidan activated autophagy by regulating the expression of autophagy markers, significantly upregulating the Bcl-2/Bax ratio, and downregulating cleaved caspase-3/caspase-3. Moreover, fucoidan improved Aβ42-induced neuronal apoptosis by activating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway and mitogen-activated protein kinase cascade in SH-SY5Y cells. Additionally, Hoechst 33342 staining showed that fucoidan diminished the level of the apoptotic nuclei in Aβ42-induced SH-SY5Ycells. Conclusion This study proposes fucoidan as a potential therapeutic candidate for AD treatment.

Modulatory Potential of LncRNA Zfas1 for Inflammation and Neuronal Apoptosis in Temporal Lobe Epilepsy

Chuan He,Caixia Su,Wentong Zhang,Qin Zhou,Xu Shen,Junjie Yang,Naixian Shi 연세대학교의과대학 2021 Yonsei medical journal Vol.62 No.3

Purpose: This study aimed to elucidate whether lncRNA ZFAS1 is involved in neuronal apoptosis and inflammation in temporal lobe epilepsy (TLE). Materials and Methods: Ninety-six TLE patients were recruited, and their peripheral venous blood was gathered to determine Zfas1 expression with polymerase chain reaction. Neurons were separated from hippocampal tissue of newborn SD rats, and siZfas1 or pcDNA3.1-Zfas1 was transfected into the neurons. Inflammatory cytokines released by neurons were determined, and neuronal activities were evaluated through MTT assay, colony formation assay, and flow cytometry. Results: Serum levels of Zfas1 were higher in TLE patients than in healthy controls (p<0.05). Furthermore, Zfas1 expression in neurons was raised by pcDNA3.1-Zfas1 and declined after silencing of Zfas1 (p<0.05). Transfection of pcDNA-Zfas1 weakened the viability and proliferation of neurons and increased neuronal apoptosis (p<0.05). Meanwhile, pcDNA3.1-Zfas1 transfection promoted lipopolysaccharide-induced release of cytokines, including tumor necrosis factor-α, interleukin (IL)-1, IL-6, and intercellular adhesion molecule-1 (p<0.05), and boosted NF-κB activation by elevating the expression of NF-κB p65, pIκBα, and IKKβ in neurons (p<0.05). Conclusion: Our results indicated that lncRNA ZFAS1 exacerbates epilepsy development by promoting neuronal apoptosis and inflammation, implying ZFAS1 as a promising treatment target for epilepsy.

Selective Suppression of a Subset of Bax-dependent Neuronal Death by a Cell Permeable Peptide Inhibitor of Bax, BIP

김수영,김현,선웅 한국통합생물학회 2008 Animal cells and systems Vol.12 No.4

Bax, a pro-apoptotic member of Bcl-2 family proteins, plays a central role in the mitochondria-dependent apoptosis. Apoptotic signals induce the translocation of Bax from cytosol into the mitochondria, which triggers the release of apoptogenic molecules such as cytochrome C and apoptosis-inducing factor, AIF. Bax-inhibiting peptide (BIP) is a cell permeable peptide comprised of five amino acids designed from the Bax-interaction domain of Ku70. Because BIP inhibits Bax translocation and Bax-mediated release of cytochrome C, BIP suppresses Bax-dependent apoptosis. In this study, we observed that BIP inhibited staurosporine-induced neuronal death in cultured cerebral cortex and cerebellar granule cells, but BIP failed to rescue granule cells from trophic signal deprivation-induced neuronal death, although both staurosporine-induced and trophic signal deprivation-induced neuronal death are dependent on Bax. These findings suggest that the mechanisms of the Bax activation may differ depending on the type of cell death induction, and thus BIP exhibits selective suppression of a subtype of Bax-dependent neuronal death. Bax, a pro-apoptotic member of Bcl-2 family proteins, plays a central role in the mitochondria-dependent apoptosis. Apoptotic signals induce the translocation of Bax from cytosol into the mitochondria, which triggers the release of apoptogenic molecules such as cytochrome C and apoptosis-inducing factor, AIF. Bax-inhibiting peptide (BIP) is a cell permeable peptide comprised of five amino acids designed from the Bax-interaction domain of Ku70. Because BIP inhibits Bax translocation and Bax-mediated release of cytochrome C, BIP suppresses Bax-dependent apoptosis. In this study, we observed that BIP inhibited staurosporine-induced neuronal death in cultured cerebral cortex and cerebellar granule cells, but BIP failed to rescue granule cells from trophic signal deprivation-induced neuronal death, although both staurosporine-induced and trophic signal deprivation-induced neuronal death are dependent on Bax. These findings suggest that the mechanisms of the Bax activation may differ depending on the type of cell death induction, and thus BIP exhibits selective suppression of a subtype of Bax-dependent neuronal death.

Hypoxia protects neuronal cells from human prion protein fragment-induced apoptosis

Seo, Jae-Suk,Seol, Jae-Won,Moon, Myung-Hee,Jeong, Jae-Kyo,Lee, You-Jin,Park, Sang-Youel Blackwell Publishing Ltd 2010 Journal of Neurochemistry Vol.112 No.3

<P><I>J. Neurochem.</I> (2010) <B>112</B>, 715–722.</P><P>Abstract</P><P>Prion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal isoform of the prion protein PrP<SUP>Sc</SUP>. Human prion protein fragment, PrP (106–126) (prion protein peptide 106–126), may contain most of the pathological features associated with PrP<SUP>Sc</SUP>. Hypoxic conditions elicit cellular responses adaptively designed to improve cell survival and have an important role in the process of cell survival. We investigate the effects of hypoxia on PrP (106–126)-induced apoptosis in the present study. Human neuroblastoma and glioblastoma cells were incubated with varied doses of PrP (106–126) under both normoxic or hypoxic conditions, in order to determine the regulatory effects of hypoxia on PrP (106–126)-induced apoptosis. The results indicate that hypoxia protects neuronal cells against PrP (106–126)-induced cell death by activating the Akt signal, which is inactivated by prion proteins, and inhibiting PrP (106–126)-induced caspase 3 activation. Low oxygen conditions increase the Bcl-2 protein, which is associated with anti-apoptotic signals, and recover the PrP (106–126)-induced reduction in mitochondrial transmembrane potential. This study demonstrates that hypoxia inhibits PrP (106–126)-induced neuron cell death by regulating Akt and Akt-related signaling, and it also suggests that prion-related neuronal damage and disease may be regulated by hypoxia or by hypoxic-inducing genes.</P>

Neuroprotective Effects of Scopoletin on Neuro-damage caused by Alcohol in Primary Hippocampal Neurons

Lee, Jina,Cho, Hyun-Jeong The Korean Society for Biomedical Laboratory Scien 2020 Biomedical Science Letters Vol.26 No.2

Excessive drinking of alcohol is known to be one of the main causes of various neurological diseases, such as Alzheimer's disease. Scopoletin is known to have anti-inflammatory and antioxidative properties, and to protect nerve cells. This study examined whether scopoletin inhibits the alcohol-induced apoptosis of primary hippocampal neurons, and how scopoletin regulates several factors associated with the caspase-mediated pathway. To achieve this, the cell viability and apoptosis rate of primary hippocampal neurons were measured by Cell Counting Kit-8 and flow cytometry, respectively. Apoptosis-related protein expressions (Bax, Bid, caspase-3, caspase-9, and Poly (ADP-ribose) polymerase (PARP)) were analyzed by Western blotting, and the ANOVA method was used to confirm the significance of the measured results. As a result, scopoletin inhibited the expressions of alcohol-induced apoptosis and apoptosis-related proteins in primary hippocampal neurons. These results suggest that down-regulation of Bid, Bax, and cleaved caspase-9 expression induced by scopoletin down-regulates the expression of cleaved caspase-3, inhibits the expression of cleaved PARP, and finally, inhibits mitochondrial apoptotic pathways. The study suggests that scopoletin is worth developing as a candidate for neuroprotective agent.

Morc2a p.S87L mutation develops peripheral and central neuropathies accompanying neuronal DNA damage and apoptosis

Geonseong Lee,Sucheong Yeom,Byungok Choi 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Microrchidia (MORC)-family CW-type zinc finger 2 (MORC2) gene is related to DNA repair, adipogenesis, lipid homeostasis, and epigenetic silencing via the human silencing hub complex. MORC2 missense mutation was known to cause Charcot-Marie-Tooth type2 Z (CMT2Z) of peripheral axonal neuropathy. However, there were reports of central neuropathy in patients with a missense mutation in the ATPase module of MORC2. It could be co-categorized by developmental delay, impaired growth, dysmorphic facies, and axonal neuropathy (DIGFAN) syndrome. The etiology of MOR2C mutation-mediated neuropathy remains uncertain. The mouse orthologue of human MORC2 is Microrchidia 2A (Morc2a). This study established Morc2a p.S87L mutant mice using CRISPR mediated single-stranded template repair and analyzed neuropathy progress and its etiology. B6.Morc2a S87L/+ animals displayed the clinical symptoms expected in human CMT2Z patients, like delayed nerve-conducting velocity, axonal neuropathy with unmyelinated axons, and locomotive dysfunction with skeletal muscle weakness. Notably, we observed severe central nervous system neuropathy with cerebella ataxia and motor neuron degeneration in the anterior horn of the spinal cord, and this seemed to be evidence of DIGFAN syndrome. B6.Morc2a S87L/+ exhibited an accumulation of DNA damage in neuronal cells, followed by p53/Cyto c/Caspase 9/Caspase 3-mediated apoptosis. Here, we present a new mouse model of CMT2Z and DIGFAN with a Morc2a p.S87L mutation. Morc2a missense mutation causes DNA damage accumulation, especially in neuronal cells, and that this is related to neuronal degeneration via apoptosis. We suggest that neuronal apoptosis is a possible target for the therapeutic approach in MORC2 missense mutation.

PKB/Akt inhibits ceramide-induced apoptosis in neuroblastoma cells by blocking apoptosis-inducing factor (AIF) translocation

Kim, Nam Hyun,Kim, Kyunghoon,Park, Weon Seo,Son, Hyeon S.,Bae, Youngmee Wiley Subscription Services, Inc., A Wiley Company 2007 Journal of cellular biochemistry Vol.102 No.5

<P>Ceramide is a sphingolipid that is abundant in the plasma membrane of neuronal cells and is thought to have regulatory roles in cell differentiation and cell death. Ceramide is known to induce apoptosis in a variety of different cell types, whereas the physiological significance of gangliosides, another class of sphingolipids, in these processes is still unclear. We examined the mechanisms of ceramide-induced cell death using a human neuroblastoma cell line. Treatment of the human neuroblastoma cell line SH-SY5Y with ceramide induced dephosphorylation of the PKB/Akt kinase and subsequent mitochondrial dysfunction. In addition, ceramide-induced neuronal cell death was not completely blocked by inhibition of caspase activity. This incomplete inhibition appeared to be attributable to the translocation of apoptosis-inducing factor to the nucleus. Furthermore, overexpression of active PKB/Akt or Bcl-2 successfully blocked ceramide-induced neuronal cell death through inhibition of the translocation of apoptosis-inducing factor. J. Cell. Biochem. 102: 1160–1170, 2007. © 2007 Wiley-Liss, Inc.</P>