[Imaging of mild cognitive impairment]. / Exploracion del deterioro cognitivo leve por neuroimagen.

The refinement of in vivo imaging approaches to investigating the structure and function of the aging brain has provided the opportunity to strengthen our knowledge of the biological substrate of normal aging and late life neurological and psychiatric disorders. While postmortem studies are biased toward the end stages of disease, functional and structural imaging have permitted us to characterize the brain changes accompanying early Alzheimer s disease (AD). As more effective therapeutic approaches to slowing (and potentially reversing) disease progression are developed, the role of imaging in determining pre AD or high risk conditions becomes increasingly important. The goal of applying non invasive means to identify the transition state of mild cognitive impairment (MCI) is of considerable public health importance. Further, emerging imaging strategies may be used to monitor the efficacy of therapeutic regimens.

Pharmacological investigation of mitochondrial ca(2+) transport in central neurons: studies with CGP-37157, an inhibitor of the mitochondrial Na(+)-Ca(2+) exchanger.

Mitochondria buffer large changes in [Ca(2+)](i)following an excitotoxic glutamate stimulus. Mitochondrial sequestration of [Ca(2+)](i)can beneficially stimulate oxidative metabolism and ATP production. However, Ca(2+)overload may have deleterious effects on mitochondrial function and cell survival, particularly Ca(2+)-dependent production of reactive oxygen species (ROS) by the mitochondria. We recently demonstrated that the mitochondrial Na(+)-Ca(2+)exchanger in neurons is selectively inhibited by CGP-37157, a benzothiazepine analogue of diltiazem. In the present series of experiments we investigated the effects of CGP-37157 on mitochondrial functions regulated by Ca(2+). Our data showed that 25 microM CGP-37157 quenches DCF fluorescence similar to 100 microM glutamate and this effect was enhanced when the two stimuli were applied together. CGP-37157 did not increase ROS generation and did not alter glutamate or 3mM hydrogen-peroxide-induced increases in ROS as measured by DHE fluorescence. CGP-37157 induces a slight decrease in intracellular pH, much less than that of glutamate. In addition, CGP-37157 does not enhance intracellular acidification induced by glutamate. Although it is possible that CGP-37157 can enhance mitochondrial respiration both by blocking Ca(2+)cycling and by elevating intramitochondrial Ca(2+), we did not observe any changes in ATP levels or toxicity either in the presence or absence of glutamate. Finally, mitochondrial Ca(2+)uptake during an excitotoxic glutamate stimulus was only slightly enhanced by inhibition of mitochondrial Ca(2+)efflux. Thus, although CGP-37157 alters mitochondrial Ca(2+)efflux in neurons, the inhibition of Na(+)-Ca(2+)exchange does not profoundly alter glutamate-mediated changes in mitochondrial function or mitochondrial Ca(2+)content.

Astrocytes are more resistant than neurons to the cytotoxic effects of increased [Zn(2+)](i).

Increased intracellular free Zn(2+) ([Zn(2+)](i)) is toxic to neurons. Glia are more resistant to Zn(2+)-mediated toxicity; however, it is not known if this is because glia are less permeable to Zn(2+) or if glia possess intrinsic mechanisms that serve to buffer or extrude excess [Zn(2+)](i). We used the Zn(2+)-selective ionophore pyrithione to directly increase [Zn(2+)](i) in both neurons and astrocytes. In neurons, a 5-min exposure to 1 microM extracellular Zn(2+) in combination with pyrithione produced widespread toxicity, whereas extensive astrocyte injury was not observed until extracellular Zn(2+) was increased to 10 microM. Measurements with magfura-2 demonstrated that pyrithione increased [Zn(2+)](i) to similar levels in both cell types. We also measured how increased [Zn(2+)](i) affects mitochondrial membrane potential (Deltapsi(m)). In astrocytes, but not in neurons, toxic [Zn(2+)](i) resulted in an acute loss of Deltapsi(m), suggesting that mitochondrial dysregulation may be an early event in [Zn(2+)](i)-induced astrocyte but not neuronal death.

Effects of oxidants and glutamate receptor activation on mitochondrial membrane potential in rat forebrain neurons.

Both glutamate and reactive oxygen species have been implicated in excitotoxic neuronal injury, and mitochondria may play a key role in the mediation of this process. In this study, we examined whether glutamate-receptor stimulation and oxidative stress interact to affect the mitochondrial membrane potential (delta psi). We measured delta psi in rat forebrain neurons with the ratiometric fluorescent dye JC-1 by using laser scanning confocal imaging. Intracellular oxidant levels were measured by using the oxidation-sensitive dyes 2',7'-dichlorodihydrofluorescein (DCFH2) and dihydroethidium (DHE). Application of hydrogen peroxide (0.3-3 mM) or 1 mM xanthine/0.06 U/ml xanthine oxidase decreased delta psi in a way that was independent of the presence of extracellular Ca2+ and was not affected by the addition of cyclosporin A, suggesting the presence of either a cyclosporin A-insensitive form of permeability transition, or a separate mechanism. tert-Butylhydroperoxide (730 microM) had less of an effect on delta psi than hydrogen peroxide despite similar effects on intracellular DCFH2 or DHE oxidation. Hydrogen peroxide-, tert-butylhydroperoxide-, and superoxide-enhanced glutamate, but not kainate, induced decreases in delta psi. The combined effect of peroxide or superoxide plus glutamate was Ca2+ dependent and was partially inhibited by cyclosporin A. These results suggest that oxidants and glutamate depolarize mitochondria by different mechanisms, and that oxidative stress may enhance glutamate-mediated mitochondrial depolarization.

Effects of pyrroloquinoline quinone on glutamate-induced production of reactive oxygen species in neurons.

Pyrroloquinoline quinone may act as a free radical scavenger and also as a modulator of the NMDA receptor associated redox modulatory site. Using the oxidation sensitive dye dihydroethidium, we examined the effects of pyrroloquinoline quinone on free radical production in cultured forebrain neurons following glutamate receptor activation. Both glutamate (100 microM) and hydrogen peroxide (30 mM) produced a rapid increase in dihydroethidium fluorescence indicating dye oxidation. Pyrroloquinoline quinone (5-200 microM) effectively inhibited dihydroethidium fluorescence induced by glutamate but not by hydrogen peroxide. Glutamate-induced dihydroethidium fluorescence was inhibited by the thiol oxidant 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). Pyrroloquinoline quinone (50 microM) inhibited glutamate responses in control and in dithiothreitol treated neurons. However, pyrroloquinoline quinone did not further decrease the response to glutamate in DTNB treated neurons. These results suggest that pyrroloquinoline quinone inhibits the free radical-generating response to glutamate by oxidizing the NMDA receptor redox site and not by scavenging reactive oxygen species.