AMPK is activated early in cerebellar granule cells undergoing apoptosis and influences VADC1 phosphorylation status and activity.

The neurodegeneration of cerebellar granule cells, after low potassium induced apoptosis, is known to be temporally divided into an early and a late phase. Voltage-dependent anion channel-1 (VDAC1) protein, changing from the closed inactive state to the active open state, is central to the switch between the early and late phase. It is also known that: (i) VDAC1 can undergo phosphorylation events and (ii) AMP-activated protein kinase (AMPK), the sensor of cellular stress, may have a role in neuronal homeostasis. In the view of this, the involvement of AMPK activation and its correlation with VDAC1 status and activity has been investigated in the course of cerebellar granule cells apoptosis. The results reported in this study show that an increased level of the phosphorylated, active, isoform of AMPK occurs in the early phase, peaks at 3 h and guarantees an increase in the phosphorylation status of VDCA1, resulting in a reduced activity of this latter. However this situation is transient in nature, since, in the late phase, AMPK activation decreases as well as the level of phosphorylated VDAC1. In a less phosphorylated status, VDAC1 fully recovers its gating activity and drives cells along the death route.

Glucose-6-phosphate tips the balance in modulating apoptosis in cerebellar granule cells.

A metabolic shift from oxidative phosphorylation to glycolysis (i.e. the Warburg effect) occurs in Alzheimer's disease accompanied by an increase of both activity and level of HK-I. The findings reported here demonstrate that in the early phase of apoptosis VDAC1 activity, but not its protein level, progressively decreases, in concomitance with the physical interaction of HK-I with VDAC1. In the late phase of apoptosis, glucose-6-phosphate accumulation in the cell causes the dissociation of the two proteins, the re-opening of the channel and the recovery of VDAC1 function, resulting in a reawakening of the mitochondrial function, thus inevitably leading to cell death.

Thioredoxin/thioredoxin reductase system involvement in cerebellar granule cell apoptosis.

The involvement of thioredoxin/thioredoxin reductase system has been investigated in cerebellar granule cells (CGCs), a cellular system in which neurons are induced in apoptosis by the physiological stimulus of lowering extracellular potassium. Clarifying the sequence of events that occur during apoptosis is a critical issue as it can lead to the identification of those key events that, if blocked, can slow down or reverse the death process. The results reported in this work show that TrxR is involved in the early phase of CGC apoptosis with an increase in activity that coincides with the increased expression of the TrxR1 isoform and guarantees the maintenance of adequate level of Trx in its reduced, active form. However, in late apoptosis, when about 50 % of cells are dead, partial proteolysis of TrxR1 by calpain occurs and the reduction of TrxR1 mRNA, together with the overall decrease in TrxR activity, contribute to increase the levels of the oxidized form of Trx. When the reduced form of Trx is externally added to apoptotic cultures, a significant reduction in cell death is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survival of CGCs.

Dissecting the molecular mechanism by which NH2htau and Aß1-42 peptides impair mitochondrial ANT-1 in Alzheimer disease.

To find out whether and how the adenine nucleotide translocator-1 (ANT-1) inhibition due to NH2htau and Aß1-42 is due to an interplay between these two Alzheimer's peptides, ROS and ANT-1 thiols, use was made of mersalyl, a reversible alkylating agent of thiol groups that are oriented toward the external hydrophilic phase, to selectively block and protect, in a reversible manner, the -SH groups of ANT-1. The rate of ATP appearance outside mitochondria was measured as the increase in NADPH absorbance which occurs, following external addition of ADP, when ATP is produced by oxidative phosphorylation and exported from mitochondria in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. We found that the mitochondrial superoxide anions, whose production is induced at the level of Complex I by externally added Aß1-42 and whose release from mitochondria is significantly reduced by the addition of the VDAC inhibitor DIDS, modify the thiol group/s present at the active site of mitochondrial ANT-1, impair ANT-1 in a mersalyl-prevented manner and abrogate the toxic effect of NH2htau on ANT-1 when Aß1-42 is already present. A molecular mechanism is proposed in which the pathological Aß-NH2htau interplay on ANT-1 in Alzheimer's neurons involves the thiol redox state of ANT-1 and the Aß1-42-induced ROS increase. This result represents an important innovation because it suggests the possibility of using various strategies to protect cells at the mitochondrial level, by stabilizing or restoring mitochondrial function or by interfering with the energy metabolism providing a promising tool for treating or preventing AD.

Helium-Neon laser irradiation of hepatocytes can trigger increase of the mitochondrial membrane potential and can stimulate c-fos expression in a Ca2+-dependent manner.

BACKGROUND AND OBJECTIVE: To gain some insight into the photostimulation of isolated hepatocytes irradiated with Helium-Neon (He-Ne) laser light certain biochemical events were studied with respect to two mechanisms: i) the direct light dependent activation of certain biochemical events investigated in intact cells and isolated mitochondria, ii) the indirect stimulation of processes per se light independent. STUDY DESIGNS/MATERIALS AND METHODS: Irradiation of either isolated hepatocytes or isolated rat liver mitochondria was carried out with He-Ne laser (wavelength, 632.8 nm; fluence, 0.24 J cm-2; fluence rate, 12 mW cm-2). Changes in mitochondrial membrane potential in isolated hepatocytes were monitored using the cationic probe safranine. The c-fos expression was studied by Northern blot and immunoblot analysis. RESULTS: As a result of irradiation, increase of the mitochondrial membrane potential was found to occur in irradiated hepatocytes both in the presence or in the absence of CaCl2. The hyperpolarization of the mitochondrial membrane is assumed to cause an increase in mitochondrial Ca2+ uptake that was measured in isolated mitochondria. Finally, an increase in c-fos expression was found in irradiated hepatocytes when incubated in the presence of CaCl2. CONCLUSION: This paper gives additional information on the mechanism by which He-Ne laser light, either directly or in a cascade-like effect dependent on increase in cell Ca2+, can cause cell stimulation.

The irradiation of hepatocytes with He-Ne laser causes an increase of cytosolic free calcium concentration and an increase of cell membrane potential, correlated with it, both increases taking place in an oscillatory manner.

Isolated hepatocytes were irradiated with Helium-Neon laser (fluence: 0.24 Joules x cm-2, fluence rate: 12 mW x cm-2) and changes of both cytosolic free Ca2+ concentration and cell membrane potential were checked by measuring fura-2 and bis-oxonol fluorescence respectively. Irradiation resulted in an enhancement in cytosolic free Ca2+ concentration that requires the presence of Ca2+ in the phase outside hepatocytes; consistently an increase in cell membrane potential was measured correlated with it. Interestingly, the rate of increase of both cytosolic free Ca2+ concentration and cell membrane potential shows special time dependent features similar to those peculiar of oscillatory processes.

Increase in cytosolic and mitochondrial protein synthesis in rat hepatocytes irradiated in vitro by He-Ne laser.

In order to gain an insight into the mechanism of cell photostimulation by laser light, protein synthesis was measured in hepatocytes irradiated with a low-power, continuous-wave He-Ne laser (fluence, 0.24 J cm(-2); fluence rate, 7 and 12 mW cm(-2)). As a result of irradiation, the rate and amount of 35S-methionine incorporated into newly synthesized proteins increased, as demonstrated by gel electrophoresis and quantitative analysis of labelled protein bands. The stimulation of protein synthesis was fluence dependent, with a maximum stimulation at 0.24 J cm(-2) for both fluence rates (12 and 7 mW cm(-2)). Both cytosolic and mitochondrial protein synthesis increased as a result of irradiation, as demonstrated by the measurement of hepatocytes previously treated with chloramphenicol and cycloheximide respectively. An initial investigation showed that stimulation of protein synthesis also occurred in hepatocytes irradiated with a non-coherent radiation source (fluence, 0.24 J cm(-2)).

Increase in <--H+/e- ratio of the cytochrome c oxidase reaction in mitochondria irradiated with helium-neon laser.

In order to gain a degree of insight into the mitochondrial component/s responsible of the mitochondria-red light interaction, isolated rat liver mitochondria were irradiated with a Helium-Neon laser (energy dose 2 Joules/cm2, light power 10 mW) and measurements made of the activity of cytochrome c oxidase. A low, but statistically significant increase in the oxygen uptake was found, as polarographically measured, in the presence of rotenone and antimycin A, with ascorbate and TMPD used as substrate pair. Measurements were also made both of the electron transfer and of proton pumping activity: as a result of a major stimulation in the proton pumping activity, about 55% increase of <--H+/e- ratio was found in irradiated mitochondria.