Antifungal, anti-inflamatory and neuritogenic activity of newly-isolated compounds from Disporopsis aspersa.

A new ester (1) and a terpenoid (2) were isolated from the dried whole plant of Disporopsis aspersa (HUA) ENGL. ex DIELS for the first time and their structures were elucidated, as well as their biological activities are described. The two compounds all showed good antifungal activities, especially furanone (2) exhibited better antifungal activity against Pseudoperonospora cubensis and Phytophthora infestans with EC50 value of 22.82, 18.90 µg/mL, respectively. Compound 1 exhibited a significant promotion on the neurite outgrowth in NGF-induced PC-12 cells, and moderate inhibition on the NO production induced by lipopolysaccharide (LPS) in BV-2 microglial cells.

Prohibitin is a positive modulator of mitochondrial function in PC12 cells under oxidative stress.

Prohibitin (PHB) is a ubiquitously expressed and evolutionarily conserved mitochondrial protein with multiple functions. We have recently shown that PHB up-regulation offers robust protection against neuronal injury in models of cerebral ischemia in vitro and in vivo, but the mechanism by which PHB affords neuroprotection remains to be elucidated. Here, we manipulated PHB expression in PC12 neural cells to investigate its impact on mitochondrial function and the mechanisms whereby it protects cells exposed to oxidative stress. PHB over-expression promoted cell survival, whereas PHB down-regulation diminished cell viability. Functionally, manipulation of PHB levels did not affect basal mitochondrial respiration, but it increased spare respiratory capacity. Moreover, PHB over-expression preserved mitochondrial respiratory function of cells exposed to oxidative stress. Preserved respiratory capacity in differentiated PHB over-expressing cells exposed to oxidative stress was associated with an elongated mitochondrial morphology, whereas PHB down-regulation enhanced fragmentation. Mitochondrial complex I oxidative degradation was attenuated by PHB over-expression and increased in PHB knockdown cells. Changes in complex I degradation were associated with alterations of respiratory chain supercomplexes. Furthermore, we showed that PHB directly interacts with cardiolipin and that down-regulation of PHB results in loss of cardiolipin in mitochondria, which may contribute to destabilizing respiratory chain supercomplexes. Taken together, these data demonstrate that PHB modulates mitochondrial integrity and bioenergetics under oxidative stress, and suggest that the protective effect of PHB is mediated by stabilization of the mitochondrial respiratory machinery and its functional capacity, by the regulation of cardiolipin content. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Parkin promotes proteasomal degradation of synaptotagmin IV by accelerating polyubiquitination.

Parkin is an E3 ubiquitin ligase whose mutations cause autosomal recessive juvenile Parkinson's disease (PD). Unlike the human phenotype, parkin knockout (KO) mice show no apparent dopamine neuron degeneration, although they demonstrate reduced expression and activity of striatal mitochondrial proteins believed to be necessary for neuronal survival. Instead, parkin-KO mice show reduced striatal evoked dopamine release, abnormal synaptic plasticity, and non-motor symptoms, all of which appear to mimic the preclinical features of Parkinson's disease. Extensive studies have screened candidate synaptic proteins responsible for reduced evoked dopamine release, and synaptotagmin XI (Syt XI), an isoform of Syt family regulating membrane trafficking, has been identified as a substrate of parkin in humans. However, its expression level is unaltered in the striatum of parkin-KO mice. Thus, the target(s) of parkin and the molecular mechanisms underlying the impaired dopamine release in parkin-KO mice remain unknown. In this study, we focused on Syt IV because of its highly homology to Syt XI, and because they share an evolutionarily conserved lack of Ca2+-binding capacity; thus, Syt IV plays an inhibitory role in Ca2+-dependent neurotransmitter release in PC12 cells and neurons in various brain regions. We found that a proteasome inhibitor increased Syt IV protein, but not Syt XI protein, in neuron-like, differentiated PC12 cells, and that parkin interacted with and polyubiquitinated Syt IV, thereby accelerating its protein turnover. Parkin overexpression selectively degraded Syt IV protein, but not Syt I protein (indispensable for Ca2+-dependent exocytosis), thus enhancing depolarization-dependent exocytosis. Furthermore, in parkin-KO mice, the level of striatal Syt IV protein was increased. Our data indicate a crucial role for parkin in the proteasomal degradation of Syt IV, and provide a potential mechanism of parkin-regulated, evoked neurotransmitter release.

Tripeptide GGH as the Inhibitor of Copper-Amyloid-ß-Mediated Redox Reaction and Toxicity.

The Aß complexes of some redox-active species, such as Cu, cause oxidative stress and induce severe toxicity by generating reactive oxygen species (ROS). Thus, Cu chelation therapy should be considered as a valuable strategy for the treatment of Alzheimer's disease (AD). However, more attention should be paid to the specific chelating ability of these chelating agents. Herein, a tripeptide GGH was used to selectively chelate the Cu(2+) in Aß-Cu complex in the presence of other metal ions (e.g., K(+), Ca(2+), Ni(2+), Mg(2+), and Zn(2+)) as shown by isothermal titration calorimetry results. GGH decreased the level of HO(•) radicals by preventing the formation of intermediate Cu(I) ion. Thus, the Cu species completely lost its catalytic activity at a superequimolar GGH/Cu(II) ratio (4:1) as observed by UV-visible spectroscopy, coumarin-3-carboxylic acid fluorescence, and BCA assay. Moreover, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay indicates that GGH increased PC-12 cell viability from 36% to 63%, and neurotoxicity partly triggered by ROS decreased. These results indicate potential development of peptide chelation therapy for AD treatment.

Chorein Sensitive Dopamine Release from Pheochromocytoma (PC12) Cells.

BACKGROUND: Chorein, a protein supporting activation of phosphoinositide 3 kinase (PI3K), participates in the regulation of actin polymerization and cell survival. A loss of function mutation of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) leads to chorea-acanthocytosis (ChAc), a neurodegenerative disorder with simultaneous erythrocyte akanthocytosis. In blood platelets chorein deficiency has been shown to compromise expression of vesicle-associated membrane protein 8 (VAMP8) and thus degranulation. The present study explored whether chorein is similarly involved in VAMP8 expression and dopamine release of pheochromocytoma (PC12) cells. METHODS: Chorein was down-regulated by silencing in PC12 cells. Transmission electron microscopy was employed to quantify the number of vesicles, RT-PCR to determine transcript levels, Western blotting to quantify protein expression and ELISA to determine dopamine release. RESULTS: Chorein silencing significantly reduced the number of vesicles, VAMP8 transcript levels and VAMP8 protein abundance. Increase of extracellular K+ from 5 mM to 40 mM resulted in marked stimulation of dopamine release, an effect significantly blunted by chorein silencing. CONCLUSIONS: Chorein deficiency down-regulates VAMP8 expression, vesicle numbers and dopamine release in pheochromocytoma cells.

Nanogrooved surface-patterns induce cellular organization and axonal outgrowth in neuron-like PC12-cells.

Modulation of a materials surface topography can be used to steer various aspects of adherent cell behaviour, such as cell directional organization. Especially nanometric sized topographies, featuring sizes similar to for instance the axons of the spiral ganglion cells, are interesting for such purpose. Here, we utilized nanosized grooves in the range of 75-500 nm, depth of 30-150 nm, and pitches between 150 nm and 1000 nm for cell culture of neuron-like PC12 cells. The organizational behaviour was evaluated after 7 days of culture by bright field and scanning electron microscopy. Nanotopographies were shown to induce aligned cell-body/axon orientation and an increased axonal outgrowth. Our findings suggest that a threshold for cell body alignment of neuronal cells exists on grooved topographies with a groove width of 130 nm, depth of 70 nm and pitch of 300 nm, while axon alignment can already be induced by grooves with 135 nm width, 52 nm depth and 200 nm pitch. However, no threshold has been found for axonal outgrowth, as all of the used patterns increased outgrowth of PC12-axons. In conclusion, surface nanopatterns have the potential to be utilized as an electrode modification for a stronger separation of cells, and can be used to direct cells towards the electrode contacts of cochlear implants.

Neuroprotective effects of Lycium chinense Miller against rotenone-induced neurotoxicity in PC12 cells.

Rotenone, an inhibitor of mitochondrial complex I, has been widely regarded as a neurotoxin because it induces a Parkinson's disease-like syndrome. The fruit and root bark of Lycium chinense Miller have been used as traditional medicines in Asia to treat neurodegenerative diseases. In this study, we examined the neuroprotective effects of Lycium chinense Miller extracts in rotenone-treated PC12 cells. Treatment with rotenone reduced PC12 cell viability and cellular ATP levels. Conversely, caspase 3/7 activity, the ratio of Bax:Bcl-2 expression levels, mitochondrial superoxide level, and intracellular calcium (Ca(2+)) concentration were elevated. Pretreatment with Lycium chinense Miller extracts significantly increased cell viability and ATP levels. Additionally, they attenuated caspase activation, mitochondrial membrane depolarization and mitochondrial superoxide production. Moreover, confocal microscopy showed that the mitochondrial staining pattern was restored from that of extracts treated cells and that the increase in intracellular Ca (2+) level was blunted by treatment with the extracts. Our results suggest that Lycium chinense Miller extracts may have the possible beneficial effects in Parkinson's disease by attenuating rotenone induced toxicity.

A subcellular analysis of genetic modulation of PINK1 on mitochondrial alterations, autophagy and cell death.

Mutations in the PTEN-induced putative kinase1 (PINK1) represent the second most frequent cause of autosomal recessive Parkinson's disease. The PINK1 protein mainly localizes to mitochondria and interacts with a variety of proteins, including the pro-autophagy protein beclin1 and the ubiquitin-ligase parkin. Upon stress conditions, PINK1 is known to recruit parkin at the surface of dysfunctional mitochondria and to activate the mitophagy cascade. Aim of this study was to use a simple and highly reproducible catecholamine cell model and transmission electron microscopy to characterize whether PINK1 could affect mitochondrial homeostasis, the recruitment of specific proteins at mitochondria, mitophagy and apoptosis. Samples were analyzed both in baseline conditions and following treatment with methamphetamine (METH), a neurotoxic compound which strongly activates autophagy and produces mitochondrial damage. Our data provide robust sub-cellular evidence that the modulation of PINK1 levels dramatically affects the morphology and number of mitochondria and the amount of cell death. In particular, especially upon METH exposure, PINK1 is able to increase the total number of mitochondria, concurrently recruit beclin1, parkin and ubiquitin and enhance the clearance of damaged mitochondria. In the absence of functional PINK1 and upon autophagy stress, we observe a failure of the autophagy system at large, with marked accumulation of dysfunctional mitochondria and dramatic increase of apoptotic cell death. These findings highlight the strong neuroprotective role of PINK1 as a key protein in the surveillance and regulation of mitochondrial homeostasis.

Involvement of GPR12 in the induction of neurite outgrowth in PC12 cells.

GPR12, an orphan G protein-coupled receptor, constitutively activates the Gs signaling pathway and further increases intracellular cyclic AMP. GPR12 overexpression has been reported to promote neurite extension in neurons or transform neuro2a neuroblastoma cells into neuron-like cells. However, the possible effects and mechanisms of GPR12 in the differentiation of PC12 cells are still unknown. The present study shows that GPR12 overexpression induced PC12 cells differentiation into neuron-like cells with enlarged cell sizes and neuritogenesis possibly via activation of Erk1/2 signaling and significantly increased the expression of several neurite outgrowth-related genes, including Bcl-xL, Bcl-2 and synaptophysin. These findings indicate that GPR12 may play a role in neurite outgrowth during PC12 cell differentiation.

Amyloid-ß peptide-induced secretion of endoplasmic reticulum chaperone glycoprotein GRP94.

Amyloid-ß (Aß) peptide-induced neurotoxicity is typically associated with cell death through mechanisms not entirely understood. Here, we investigated stress signaling events triggered by soluble Aß in differentiated rat neuronal-like PC12 cells. Morphologic evaluation of apoptosis confirmed that Aß induced nuclear fragmentation that was prevented by pre-treatment with the antiapoptotic bile acid tauroursodeoxycholic acid (TUDCA). In addition, Aß exposure triggered an early signaling response by the endoplasmic reticulum (ER) and caspase-12-mediated apoptosis, which, however, was independent of the ER-stress pathway. Furthermore, ER stress markers, including GRP94, ATF-6α, CHOP, and eIF2α, were strongly downregulated by Aß, independent of protein degradation, and partially restored by TUDCA. Calpain inhibition prevented caspase-12 activation and reduced levels of ATF-6α. Importantly, Aß-induced GRP94 downregulation was related to protein secretion and partially rescued through inhibition of the secretory pathway by geldanamycin and brefeldin. In conclusion, we showed that the ER is a proximal stress sensor for soluble Aß-induced toxicity, resulting in caspase-12 activation and cell death in PC12 neuronal cells. Moreover, ER chaperone GRP94 secretion was associated with Aß-induced apoptotic signaling. These data provide new information linking apoptotic properties of Aß peptide to distinct subcellular mechanisms of toxicity. Further characterization of this signaling pathway is likely to provide new perspectives for modulation of amyloid-induced apoptosis.

Aucubin modulates Bcl-2 family proteins expression and inhibits caspases cascade in H2O2-induced PC12 cells.

In this study, the effect of aucubin on H(2)O(2)-induced apoptosis was studied by using a rat pheochromocytoma (PC12) cell line. We have analyzed the apoptosis of H(2)O(2)-induced PC12 cells, H(2)O(2)-induced apoptosis appeared to correlate with lower Bcl-2 expression, higher Bax expression and sequential activation of caspase-3 leading to cleavage of poly-ADP-ribose polymerase (PARP). Aucubin not only inhibited lower Bcl-2 expression, high Bax expression, but also modulated caspase-3 activation, PARP cleavage, and eventually protected against H(2)O(2)-induced apoptosis. These results indicated that aucubin can obstruct H(2)O(2)-induced apoptosis by regulating of the expression of Bcl-2 and Bax, as well as suppression of caspases cascade activation.

Neurites from PC12 cells are connected to each other by synapse-like structures.

PC12 cells have been used as a model of sympathetic neurons. Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and cAMP induce neurite outgrowth from PC12 cells. cAMP induced a greater number of neurites than did NGF. In particular, we attempted to elucidate whether PC12 cell neurites, induced by several factors including NGF, bFGF, and cAMP, form synapses, and whether each neurite has presynaptic and postsynaptic properties. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), we observed that neurites are connected to each other. The connected regions presented dense core vesicles and a clathrin-coated membrane invagination. In addition, typical maker proteins for axon and dendrite were identified by an immuno-staining method. Tau-1, an axonal marker in neurons, was localized at a high concentration in the terminal tips of neurites from PC12 cells, which were connected to neurite processes containing MAP-2, a dendritic marker in neurons. Furthermore, neurites containing SV2 and synaptotagmin, markers of synaptic vesicles, were in contact with neurites harboring drebrin, a marker of the postsynaptic membrane, suggesting that neurites from PC12 cells induced by NGF, bFGF, and cAMP may form synapse-like structures. Tat-C3 toxin, a Rho inhibitor, augmented neurite outgrowth induced by NGF, bFGF, and cAMP. Tat-C3 toxin together with neurotrophins also exhibited synapse-like structures between neurites. However, it remains to be studied whether RhoA inhibition plays a role in the formation of synapse-like structures in PC12 cells.

Nerve growth factor attenuates 2-deoxy-d-glucose-triggered endoplasmic reticulum stress-mediated apoptosis via enhanced expression of GRP78.

The glucose analog 2-deoxy-d-glucose (2DG) depletes cells of glucose. Inhibition of glycosylation caused by glucose depletion induces endoplasmic reticulum (ER) stress with subsequent apoptosis. Glucose-regulated protein 78 (GRP78) is a molecular chaperone that acts within the ER. During ER stress, GRP78 expression is induced as part of the unfolded protein response (UPR). We found that nerve growth factor (NGF) prevented 2DG-triggered ER stress-mediated apoptosis, but not the induction of GRP78 expression, in PC12 cells. Surprisingly, GRP78 expression was further up-regulated when NGF was added to 2DG-treated PC12 cells. When a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K), LY294002, was added to 2DG plus NGF-treated cells, both the effects of NGF on 2DG-induced apoptosis and GRP78 expression were significantly diminished. In addition, versipelostatin (VST), a specific inhibitor of GRP78 expression, and small interfering RNA (siRNA) against GRP78 mRNA also decreased both the effects of NGF on 2DG-induced apoptosis and GRP78 expression. RT-PCR and Western blot analyses revealed that enhanced production of nuclear p50 ATF6, but not spliced XBP1, mainly contributed to the NGF-induced enhancement of GRP78 expression in 2DG-treated cells. These results suggest that the NGF-activated PI3-K/Akt signaling pathway plays a protective role against ER stress-mediated apoptosis via enhanced expression of GRP78 in PC12 cells.

Alzheimer's disease genetic mutation evokes ultrastructural alterations: correlation to an intracellular Abeta deposition and the level of GSK-3beta-P(Y216) phosphorylated form.

Herein we demonstrate that PC12 cells, which overexpress human wild-type amyloid-beta precursor protein (AbetaPPwt) or AbetaPP bearing double Swedish mutation (AbetaPPsw), reveal phenotype characteristic for Alzheimer's disease (AD). The examination of cell ultrastructure showed the presence of peptide aggregates within the cells, activation of endosomal-lysosomal system and extensive exocytosis. Furthermore, the autophagy induction was also characteristic hallmark of amyloid-beta-induced cytotoxicity. Morphological changes were positively correlated with the extent of phosphorylated glycogen synthase kinase-3beta (phospho-Tyr(216)-GSK-3beta, GSK-3beta-P(Y216)). The activity of GSK-3beta is believed to cause tau protein hyper-phosphorylation, increased amyloid-beta production and local plaque-associated microglial-mediated inflammatory responses. All of them are symptomatic for AD. In our studies, the highly significant Y216 phosphorylation and over-expression of total GSK-3beta were observed in AbetaPPsw-transfected PC12 cells. In addition, the immuocytochemical analysis showed co-localization of GSK-3beta-P(Y216) and amyloid-beta deposits. Thus, our data support a functional role of GSK-3beta in AbetaPP processing, further implicating this kinase in the amyloid-beta-dependent pathogenesis.

3,4-Dihydroxyphenylacetic acid (DOPAC) modulates the toxicity induced by nitric oxide in PC-12 cells via mitochondrial dysfunctioning.

It has been postulated that dihydroxyphenylacetic acid (DOPAC), a major dopamine metabolite, and nitric oxide (NO) induce mitochondrial dysfunction in a synergistic manner. We examined the combined effects of NO and DOPAC on PC-12 cells in terms of cell viability, nuclear morphology, mitochondrial parameters and cell death mechanisms. The apoptotic cell death induced by the NO-donor, S-nitroso-N-acetylpenicillamine (SNAP), was differently modulated by DOPAC as a function of DOPAC/cell ratios. Whereas below 200nmol/10(6) cells, DOPAC inhibited a typical apoptotic pathway induced by exposure the cells to the NO donor, above 200nmol DOPAC/10(6) cells, the cell death was not only enhanced but encompassed a distinct mechanism. Loading the cells with dopamine mimicked the effects of DOPAC. Specifically, the combination of DOPAC and NO induced an early mitochondrial membrane potential dissipation and ATP depletion followed by loss of cellular membrane integrity. Mitochondrial dysfunction was accompanied by the release of cytochrome c in both cases, NO individually and in combination with DOPAC, but caspase-3 and caspase-9 activation were only observed in the absence of DOPAC. DOPAC alone was ineffective. Thus, our results suggest a role for DOPAC as a modulator of cell fate and point to a pathway of cell death involving DOPAC and NO, via mechanisms that include mitochondrial dysfunction but do not involve the activation of the typical apoptotic caspase cascade. The significance of these results is discussed in connection with the mechanisms of cell death underlying Parkinson's disease.

Syntaxin 6 regulates nerve growth factor-dependent neurite outgrowth.

Neurite outgrowth is crucial for neural circuit formation. Intracellular membrane trafficking is involved in the cell surface expansion that is necessary for neurite outgrowth. It is known that syntaxin 6 is predominantly located in the Golgi region in undifferentiated PC12 cells and that it regulates trans-Golgi network trafficking and the secretory pathway via its coiled-coil domains. However, whether it also regulates neurite outgrowth remains unknown. In this paper, we found that syntaxin 6 was located both in the Golgi apparatus and the distal tips of the neurites of nerve growth factor (NGF)-treated PC12 cells. We also showed that the overexpression of the first coiled-coil domain of syntaxin 6 inhibited NGF-dependent neurite outgrowth. However, the coiled-coil domain-disrupting mutant had little effect on neurite outgrowth. These results suggest that the first coiled-coil domain of syntaxin 6 plays a crucial role in NGF-dependent neurite outgrowth.

Phospholipid mediated plasticity in exocytosis observed in PC12 cells.

Membrane composition serves to identify intracellular compartments, signal cell death, as well as to alter a cell's electrical and physical properties. Here we use amperometry to show that supplementation with the phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and phosphatidylserine (PS) can alter several aspects of exocytosis. Changes in the amperometric peak shape derived from individual exocytosing vesicles reveal that PC slows expulsion of neurotransmitter while PE accelerates expulsion of neurotransmitter. Amperometry data reveal a reduced amount of catecholamine released per event from PC-treated cells while electron micrographs indicate the vesicles in these cells are 50% larger than controls, thus providing evidence of pharmacological changes in vesicle concentration. Addition of SM appears to affect the rate of fusion pore expansion, indicated by slower peak rise times, but does not affect decay times or quantal size. Addition of PS results in a 1.7-fold increase in the number of events elicited by high-K(+) depolarization. Electron micrographs of PS-treated cells suggest that increased vesicle recruitment underlies enhanced secretion. We did not observe any effect of phosphatidylinositol (PI) treatment. Together these data suggest that differences in membrane composition affect exocytosis and might be involved in mechanisms of cell function controlling the dynamics of communication via exocytosis.

The calcium-sensing protein synaptotagmin 7 is expressed on different endosomal compartments in endocrine, neuroendocrine cells or neurons but not on large dense core vesicles.

Synaptotagmin (syt) isoforms function as calcium sensor in post-Golgi transport although the precise transport step and compartment(s) concerned are still not fully resolved. As syt7 has been proposed to operate in lysosomal exocytosis and in exocytosis of large dense core vesicles (LDCVs), we have addressed the distribution of endogenous syt7 in insulin-secreting cells. These cells express different syt7 isoforms comparable to neurons. According to subcellular fractionation and quantitative confocal immunocytochemistry, syt7 is not found on LDCVs or on synaptic-like microvesicles but colocalizes with Rab7 on endosomes and to structures near to or at the plasma membrane. Similarly, endogenous syt7 was absent from LDCVs in pheochromocytoma PC12 cells. In contrast, syt7 localised to lysosomes in both, PC12 cells and hippocampal neurons. In conclusion, endogenous syt7 shows a wider distribution than previously reported but does not qualify as vesicular calcium sensor in SLMV or LDCV exocytosis according to its localisation.