Preliminary Study on Clusterin Protein (sCLU) Expression in PC-12 Cells Overexpressing Wild-Type and Mutated (Swedish) AβPP genes Affected by Non-Steroid Isoprenoids and Water-Soluble Cholesterol

In this study we attempted to verify the hypothesis that the mevalonate pathway affects amyloid beta precursor protein (AβPP) processing and regulates clusterin protein levels. AβPP expression was monitored by green fluorescence (FL) and Western blot (WB). WB showed soluble amyloid protein precursor alpha (sAβPPα) presence in AβPP-wt cells and Aβ expression in AβPP-sw cells. Nerve growth factor (NGF)-differentiated rat neuronal pheochromocytoma PC-12 cells were untreated/treated with statins alone or together with non-sterol isoprenoids. Co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol demonstrated statin-dependent neurotoxicity resulted from the attenuated activity of mevalonate pathway rather than lower cholesterol level. Atorvastatin (50 μM) or simvastatin (50 μM) as well as cholesterol chelator methyl-β-cyclodextrin (0.2 mM) diminished cell viability (p < 0.05) and clusterin levels. Interestingly, co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol stimulated (p < 0.05) clusterin expression. Effects of non-sterol isoprenoids, but not water soluble cholesterol (Chol-PEG), were the most significant in mock-transfected cells. Geranylgeraniol (GGOH) overcame atorvastatin (ATR)-dependent cytotoxicity. This effect does not seem to be dependent on clusterin, as its level became lower after GGOH. The novelty of these findings is that they show that the mevalonate (MEV) pathway rather than cholesterol itself plays an important role in clusterin expression levels. In mock-transfected, rather than in AβPP-overexpressing cells, GGOH/farnesol (FOH) exerted a protective effect. Thus, protein prenylation with GGOH/FOH might play substantial role in neuronal cell survival.

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Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency

Cellular Physiology and Biochemistry ◽

10.1159/000471235 ◽

2017 ◽

Vol 41 (4) ◽

pp. 1649-1660 ◽

Cited By ~ 7

Author(s):

Paola Maura Tricarico ◽

Alessandra Romeo ◽

Rossella Gratton ◽

Sergio Crovella ◽

Fulvio Celsi

Keyword(s):

Cell Death ◽

Mevalonate Pathway ◽

Cell Model ◽

Neuroblastoma Cell ◽

Neuronal Cell ◽

Autophagic Flux ◽

Mevalonate Kinase Deficiency ◽

Mevalonate Kinase ◽

Protein Prenylation ◽

Prenylated Proteins

Background/Aims: Mevalonate Kinase Deficiency (MKD), is a hereditary disease due to mutations in mevalonate kinase gene (MVK). MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA), the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. Methods: SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. Results: MVK mutants’ over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor) further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. Conclusions: We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation.

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Expression of Apoptosis-Related Genes in Cat Testicular Tissue in Relation to Sperm Morphology and Seasonality—A Preliminary Study

Animals ◽

10.3390/ani11020489 ◽

2021 ◽

Vol 11 (2) ◽

pp. 489

Author(s):

Sylwia Prochowska ◽

Agnieszka Partyka ◽

Wojciech Niżański

Keyword(s):

Sperm Morphology ◽

Semen Quality ◽

Testicular Tissue ◽

Reproductive Season ◽

Tissue Samples ◽

Protein Levels ◽

Expression Of Genes ◽

Testicular Regression ◽

Preliminary Study ◽

Apoptotic Genes

Apoptosis is a crucial process in spermatogenesis, responsible for the elimination of abnormal sperm cells and testicular regression out of breeding season. The aim of this study was to assess if the expression of apoptosis-related genes in testicular tissue of domestic cats differed: (1) between normozoospermic and teratozoospermic donors, and (2) between reproductive and non-reproductive season. The expression of genes: BCL2L1, BCL2, BAX, BAD, FAS, FASLG, and caspases (CASP3, CASP8, CASP9, and CASP10) was analyzed by qRT-PCR in testicular tissue samples. During non-reproductive season significantly higher expression of two anti-apoptotic genes (BCL2L1 and BCL2) was observed. Additionally, there was a significant higher expression of CASP10 in teratozoospermic cats during non-reproductive than during reproductive season. No differences were noted between normozoospermic and teratozoospermic groups. Upregulation of some genes during the non-reproductive season indicates engagement of apoptotic mechanisms in the seasonal changes of semen quality in cats, however further studies on protein levels and analysis of changes on distinct testicular germinal layers are required. At the same time, teratozoospermia in the general population of cats seems to be not connected with dysregulation of apoptosis in the testes.

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MitoQ Is Able to Modulate Apoptosis and Inflammation

International Journal of Molecular Sciences ◽

10.3390/ijms22094753 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4753

Author(s):

Elisa Piscianz ◽

Alessandra Tesser ◽

Erika Rimondi ◽

Elisabetta Melloni ◽

Claudio Celeghini ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Mevalonate Pathway ◽

Neuronal Cell ◽

Reactive Oxygen ◽

Mitochondrial Activity ◽

Oxygen Species ◽

Short Period ◽

Inflammatory Stimuli ◽

Apoptosis And Inflammation ◽

The Impact

Mitoquinone (MitoQ) is a mitochondrial reactive oxygen species scavenger that is characterized by high bioavailability. Prior studies have demonstrated its neuroprotective potential. Indeed, the release of reactive oxygen species due to damage to mitochondrial components plays a pivotal role in the pathogenesis of several neurodegenerative diseases. The present study aimed to examine the impact of the inflammation platform activation on the neuronal cell line (DAOY) treated with specific inflammatory stimuli and whether MitoQ addition can modulate these deregulations. DAOY cells were pre-treated with MitoQ and then stimulated by a blockade of the cholesterol pathway, also called mevalonate pathway, using a statin, mimicking cholesterol deregulation, a common parameter present in some neurodegenerative and autoinflammatory diseases. To verify the role played by MitoQ, we examined the expression of genes involved in the inflammation mechanism and the mitochondrial activity at different time points. In this experimental design, MitoQ showed a protective effect against the blockade of the mevalonate pathway in a short period (12 h) but did not persist for a long time (24 and 48 h). The results obtained highlight the anti-inflammatory properties of MitoQ and open the question about its application as an effective adjuvant for the treatment of the autoinflammatory disease characterized by a cholesterol deregulation pathway that involves mitochondrial homeostasis.

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Insertion of the βGeo Promoter Trap into the Fem1c Gene of ROSA3 Mice

Molecular and Cellular Biology ◽

10.1128/mcb.24.9.3794-3803.2004 ◽

2004 ◽

Vol 24 (9) ◽

pp. 3794-3803 ◽

Cited By ~ 6

Author(s):

Cassandra L. Schlamp ◽

Andrew T. Thliveris ◽

Yan Li ◽

Louis P. Kohl ◽

Claudia Knop ◽

...

Keyword(s):

Cell Death ◽

Ganglion Cells ◽

Neuronal Cell Death ◽

Insertion Site ◽

Neuronal Cell ◽

Pyramidal Cells ◽

Coding Region ◽

Protein Levels ◽

Molecular Events ◽

Promoter Trap

ABSTRACT ROSA3 mice were developed by retroviral insertion of the βGeo gene trap vector. Adult ROSA3 mice exhibit widespread expression of the trap gene in epithelial cells found in most organs. In the central nervous system the highest expression of βGeo is found in CA1 pyramidal cells of the hippocampus, Purkinje cells of the cerebellum, and ganglion cells of the retina. Characterization of the genomic insertion site for βGeo in ROSA3 mice shows that the trap vector is located in the first intron of Fem1c, a gene homologous to the sex-determining gene fem-1 of Caenorhabditis elegans. Transcription of the Rosa3 allele (R3) yields a spliced message that includes the first exon of Fem1c and the βGeo coding region. Although normal processing of the Fem1c transcript is disrupted in homozygous Rosa3 (Fem1cR3/R3 ) mice, some tissues show low levels of a partially processed transcript containing exons 2 and 3. Since the entire coding region of Fem1c is located in these two exons, Fem1cR3/R3 mice may still be able to express a putative FEM1C protein. To this extent, Fem1cR3/R3 mice show no adverse effects in their sexual development or fertility or in the attenuation of neuronal cell death, another function that has been attributed to both fem-1 and a second mouse homolog, Fem1b. Examination of βGeo expression in ganglion cells after exposure to damaging stimuli indicates that protein levels are rapidly depleted prior to cell death, making the βGeo reporter gene a potentially useful marker to study early molecular events in damaged neurons.

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Inhibition of protein geranylgeranylation induces apoptosis in myeloma plasma cells by reducing Mcl-1 protein levels

Blood ◽

10.1182/blood-2003-03-0970 ◽

2003 ◽

Vol 102 (9) ◽

pp. 3354-3362 ◽

Cited By ~ 85

Author(s):

Niels W. C. J. van de Donk ◽

Marloes M. J. Kamphuis ◽

Berris van Kessel ◽

Henk M. Lokhorst ◽

Andries C. Bloem

Keyword(s):

Multiple Myeloma ◽

Plasma Cells ◽

Mevalonate Pathway ◽

Cytochrome C Release ◽

Hmg Coa Reductase ◽

Myeloma Cells ◽

Protein Levels ◽

Geranylgeranyl Transferase ◽

Induced Apoptosis ◽

Coa Reductase

AbstractHMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway leading to the formation of cholesterol and isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The inhibition of HMG-CoA reductase by lovastatin induced apoptosis in plasma cell lines and tumor cells from patients with multiple myeloma. Here we show that cotreatment with mevalonate or geranylgeranyl moieties, but not farnesyl groups, rescued myeloma cells from lovastatin-induced apoptosis. In addition, the inhibition of geranylgeranylation by specific inhibition of geranylgeranyl transferase I (GGTase I) induced the apoptosis of myeloma cells. Apoptosis triggered by the inhibition of geranylgeranylation was associated with reduction of Mcl-1 protein expression, collapse of the mitochondrial transmembrane potential, expression of the mitochondrial membrane protein 7A6, cytochrome c release from mitochondria into the cytosol, and stimulation of caspase-3 activity. These results imply that protein geranylgeranylation is critical for regulating myeloma tumor cell survival, possibly through regulating Mcl-1 expression. Our results show that pharmacologic agents such as lovastatin or GGTase inhibitors may be useful in the treatment of multiple myeloma.

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Formation of Water-Soluble Fullerenes [C60,C70] under Ultrasonication and Antioxidant Effect

Eurasian Chemico-Technological Journal ◽

10.18321/ectj591 ◽

2017 ◽

Vol 5 (1) ◽

pp. 61

Author(s):

Weon-Bae Ko ◽

Hong-Seok Jeong ◽

Sung-Ho Hwang

Keyword(s):

Hydrogen Peroxide ◽

Room Temperature ◽

Antioxidant Effect ◽

Water Soluble ◽

Maldi Tof Ms ◽

Pc 12 Cells ◽

Effect Of Water ◽

Maldi Tof ◽

Line Following ◽

Tof Ms

The water-soluble fullerenes [C60, C70] are prepared with fullerenes [C60, C70] and a mixture of oxidants (v/v) at the ratio of 3:1 under ultrasonic condition at room temperature. The MALDI-TOF MS confirmed that the water-soluble compounds were C60 and C70. The antioxidant effect of water-soluble fullerenes [C60, C70] in the PC 12 cells (Rat pheochromocytoma) line following exposure to hydrogen peroxide (H2O2) was investigated.

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Preliminary study of an esterase-susceptible water-soluble agent (S-73) for regional chemotherapy

Cancer ◽

10.1002/1097-0142(1967)20:5<826::aid-cncr2820200539>3.0.co;2-z ◽

1967 ◽

Vol 20 (5) ◽

pp. 826-828 ◽

Cited By ~ 3

Author(s):

Louis E. Goodman ◽

Adolph Ulfohn ◽

Stanley P. Kramer ◽

Simon Calle ◽

Daniel Bakal ◽

...

Keyword(s):

Regional Chemotherapy ◽

Water Soluble ◽

Preliminary Study

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An Autophagy Modifier Screen Identifies Small Molecules Capable of Reducing Autophagosome Accumulation in a Model of CLN3-Mediated Neurodegeneration

Cells ◽

10.3390/cells8121531 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1531 ◽

Cited By ~ 5

Author(s):

Anton Petcherski ◽

Uma Chandrachud ◽

Elisabeth Butz ◽

Madeleine Klein ◽

Wen-Ning Zhao ◽

...

Keyword(s):

Protein Function ◽

Mevalonate Pathway ◽

Cell Model ◽

Major Gene ◽

Neuronal Cell ◽

Mitochondrial Atpase ◽

Lysosomal Storage ◽

Storage Material ◽

Cln3 Disease ◽

Cellular Components

Alterations in the autophagosomal–lysosomal pathway are a major pathophysiological feature of CLN3 disease, which is the most common form of childhood-onset neurodegeneration. Accumulating autofluorescent lysosomal storage material in CLN3 disease, consisting of dolichols, lipids, biometals, and a protein that normally resides in the mitochondria, subunit c of the mitochondrial ATPase, provides evidence that autophagosomal–lysosomal turnover of cellular components is disrupted upon loss of CLN3 protein function. Using a murine neuronal cell model of the disease, which accurately mimics the major gene defect and the hallmark features of CLN3 disease, we conducted an unbiased search for modifiers of autophagy, extending previous work by further optimizing a GFP-LC3 based assay and performing a high-content screen on a library of ~2000 bioactive compounds. Here we corroborate our earlier screening results and identify expanded, independent sets of autophagy modifiers that increase or decrease the accumulation of autophagosomes in the CLN3 disease cells, highlighting several pathways of interest, including the regulation of calcium signaling, microtubule dynamics, and the mevalonate pathway. Follow-up analysis on fluspirilene, nicardipine, and verapamil, in particular, confirmed activity in reducing GFP-LC3 vesicle burden, while also demonstrating activity in normalizing lysosomal positioning and, for verapamil, in promoting storage material clearance in CLN3 disease neuronal cells. This study demonstrates the potential for cell-based screening studies to identify candidate molecules and pathways for further work to understand CLN3 disease pathogenesis and in drug development efforts.

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ATP Receptor-Operated Ca2+ Influx and Catecholamine Release From Neuronal Cells

Physiology ◽

10.1152/physiologyonline.1992.7.2.56 ◽

1992 ◽

Vol 7 (2) ◽

pp. 56-59 ◽

Cited By ~ 1

Author(s):

K Inoue ◽

K Nakazawa

Keyword(s):

Mechanism Of Action ◽

Physiological Function ◽

Neuronal Cells ◽

Neuronal Cell ◽

Extracellular Atp ◽

Catecholamine Release ◽

Cation Channels ◽

Pc 12 Cells ◽

P2 Purinoceptors

The physiological function of extracellular ATP was investigated using PC-12 cells as a model neuronal cell. The mechanism of action of ATP proposed is that ATP stimulates P2-purinoceptors, activates Ca2+-permeable cation channels, and causes Ca2+ influx, triggering catecholamine release.

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Thermoresponsive Poly(N-Isopropylacrylamide-co-Dimethylaminoethyl Methacrylate) Microgel Aqueous Dispersions with Potential Antimicrobial Properties

Polymers ◽

10.3390/polym11040606 ◽

2019 ◽

Vol 11 (4) ◽

pp. 606 ◽

Cited By ~ 6

Author(s):

Coro Echeverría ◽

Alejandro Aragón-Gutiérrez ◽

Marta Fernández-García ◽

Alexandra Muñoz-Bonilla ◽

Daniel López

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Agents ◽

Antimicrobial Properties ◽

Viscoelastic Behavior ◽

Water Soluble ◽

Alkylation Reaction ◽

Aqueous Dispersions ◽

Dimethylaminoethyl Methacrylate ◽

Preliminary Study ◽

Potential Antimicrobial Activity

The work herein describes the preparation of thermoresponsive microgels with potential antimicrobial properties. Most of the work performed so far regarding microgels with antimicrobial activity, deals with the ability of microgels to carry and release antibiotics or antimicrobial agents (antimicrobial peptides). The originality of this work lies in the possibility of developing intrinsic antimicrobial microgels by copolymerization of the well-known thermoresponsive monomer, N-isopropylacrylamide (NIPAM) with dimethylaminoethyl methacrylate (DMAEMA), a water-soluble monomer, to form microgels via precipitation polymerization (radical polymerization). Due to the presence of a tertiary amine in the DMAEMA comonomer, microgels can be modified by N-alkylation reaction with methyl and butyl iodide. This quaternization confers positive charges to the microgel surfaces and thus the potential antimicrobial activity. The effect of DMAEMA content and its quaternization with both, methyl and butyl iodide is evaluated in terms of thermal and surface charge properties, as well as in the microgel size and viscoelastic behavior. Finally, a preliminary study of the antimicrobial activity against different microorganisms is also performed in terms of minimum inhibitory concentration (MIC). From this study we determined that in contrast with butylated microgels, methylated ones show potential antimicrobial activity and good physical properties besides of maintaining microgel thermo-responsiveness.

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