Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons

Developing neuronal populations undergo significant attrition by natural cell death. Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis during synaptogenesis. Following this time window, destruction of the anatomic target of dopaminergic neurons results in dopaminergic cell death but the morphology is no longer apoptotic. We describe ultrastructural changes that appear unique to dying embryonic dopaminergic neurons. In primary cultures of mesencephalon, death of dopaminergic neurons is triggered by activation of glutamate receptors sensitive to alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA), and differs ultrastructurally from both neuronal apoptosis or typical excitotoxicity. AMPA causes morphological changes selectively in dopaminergic neurons, without affecting other neurons in the same culture dishes. Two hours after the onset of treatment swelling of Golgi complexes is apparent. At 3 h, dopaminergic neurons display loss of membrane asymmetry (coinciding with commitment to die), as well as nuclear membrane invagination, irregular aggregation of chromatin, and mitochondrial swelling. Nuclear changes continue to worsen until loss of cytoplasmic structures and cell death begins to occur after 12 h. These changes are different from those described in neurons undergoing either apoptosis or excitotoxic death, but are similar to ultrastructural changes observed in spontaneous death of dopaminergic neurons in the natural mutant weaver mouse.Fil: Dorsey, D. A.. Washington University in St. Louis; Estados UnidosFil: Masco, Daniel Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Washington University in St. Louis; Estados Unidos. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; ArgentinaFil: Dikranian, K.. Washington University in St. Louis; Estados UnidosFil: Hyrc, K.. Washington University in St. Louis; Estados UnidosFil: Masciotra, L.. Washington University in St. Louis; Estados UnidosFil: Faddis, B.. Washington University in St. Louis; Estados UnidosFil: Soriano, M.. Universidad de Valencia; EspañaFil: Gru, A. A.. Washington University in St. Louis; Estados UnidosFil: Goldberg, M. P.. Universidad de Valencia; España. Washington University in St. Louis; Estados UnidosFil: de Erausquin, Gabriel Alejandro. Washington University in St. Louis; Estados Unido

Mitochondria and calcium: from cell signalling to cell death

While a pathway for Ca2+ accumulation into mitochondria has long been established, its functional significance is only now becoming clear in relation to cell physiology and pathophysiology. The observation that mitochondria take up Ca2+ during physiological Ca2+ signalling in a variety of cell types leads to four questions: (i) ‘What is the impact of mitochondrial Ca2+ uptake on mitochondrial function?’ (ii) ‘What is the impact of mitochondrial Ca2+ uptake on Ca2+ signalling?’ (iii) ‘What are the consequences of impaired mitochondrial Ca2+ uptake for cell function?’ and finally (iv) ‘What are the consequences of pathological [Ca2+]c signalling for mitochondrial function?’ These will be addressed in turn. Thus: (i) accumulation of Ca2+ into mitochondria regulates mitochondrial metabolism and causes a transient depolarisation of mitochondrial membrane potential. (ii) Mitochondria may act as a spatial Ca2+ buffer in many cells, regulating the local Ca2+ concentration in cellular microdomains. This process regulates processes dependent on local cytoplasmic Ca2+ concentration ([Ca2+]c), particularly the flux of Ca2+ through IP3-gated channels of the endoplasmic reticulum (ER) and the channels mediating capacitative Ca2+ influx through the plasma membrane. Consequently, mitochondrial Ca2+ uptake plays a substantial role in shaping [Ca2+]c signals in many cell types. (iii) Impaired mitochondrial Ca2+ uptake alters the spatiotemporal characteristics of cellular [Ca2+]c signalling and downregulates mitochondrial metabolism. (iv) Under pathological conditions of cellular [Ca2+]c overload, particularly in association with oxidative stress, mitochondrial Ca2+ uptake may trigger pathological states that lead to cell death. In the model of glutamate excitotoxicity, microdomains of [Ca2+]c are apparently central, as the pathway to cell death seems to require the local activation of neuronal nitric oxide synthase (nNOS), itself held by scaffolding proteins in close association with the NMDA receptor. Mitochondrial Ca2+ uptake in combination with NO production triggers the collapse of mitochondrial membrane potential, culminating in delayed cell death

Quantitative evaluation of mitochondrial calcium content in rat cortical neurones following a glutamate stimulus

Recent observations showed that a mitochondrial Ca2+ increase is necessary for an NMDA receptor stimulus to be toxic to cortical neurones. In an attempt to determine the magnitude of the Ca2+ fluxes involved in this phenomenon, we used carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial proton gradient uncoupler, to release mitochondrial free calcium ([Ca2+]m) during and following a glutamate stimulus, and magfura-2 to monitor cytoplasmic free calcium ([Ca2+]c).FCCP treatment of previously unstimulated neurones barely changed [Ca2+]c whereas when added after a glutamate stimulus it elevated [Ca2+]c to a much greater extent than did exposure to glutamate, suggesting a very large accumulation of Ca2+ in the mitochondria.Mitochondrial Ca2+ uptake was dependent on glutamate concentration, whereas the changes in the overall quantity of Ca2+ entering the cell, obtained by simultaneously treating neurones with glutamate and FCCP, showed a response that was essentially all-or-none.Mitochondrial Ca2+ uptake was also dependent on the nature and duration of a given stimulus as shown by comparing [Ca2+]m associated with depolarization and treatment with kainate, NMDA or glutamate. Large mitochondrial Ca2+ accumulation only occurred after a glutamate or NMDA stimulus.These studies provide a method of estimating the accumulation of Ca2+ in the mitochondria of neurones, and suggest that millimolar concentrations of Ca2+ may be reached following intense glutamate stimulation. It was shown that substantially more Ca2+ enters neurones following glutamate receptor activation than is reflected by [Ca2+]c increases

Excitotoxic mitochondrial depolarisation requires both calcium and nitric oxide in rat hippocampal neurons

Glutamate neurotoxicity has been attributed to cellular Ca2+ overload. As mitochondrial depolarisation may represent a pivotal step in the progression to cell death, we have used digital imaging techniques to examine the relationship between cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial potential (ΔΨm) during glutamate toxicity, and to define the mechanisms underlying mitochondrial dysfunction.In cells of > 11 days in vitro (DIV), exposure to 50 mM potassium or 100 μM glutamate had different consequences for ΔΨm. KCl caused a small transient loss of ΔΨm but in response to glutamate there was a profound loss of ΔΨm. In cells of 7–10 DIV, glutamate caused only a modest and reversible drop in ΔΨm.Using fura-2 to measure [Ca2+]c, responses to KCl and glutamate did not appear significantly different. However, use of the low affinity indicator fura-2FF revealed a difference in the [Ca2+]c responses to KCl and glutamate, which clearly correlated with the loss of ΔΨm. Neurons exhibiting a profound mitochondrial depolarisation also showed a large secondary increase in the fura-2FF ratio.The glutamate-induced loss of ΔΨm was dependent on Ca2+ influx. However, inhibition of nitric oxide synthase (NOS) by L-NAME significantly attenuated the loss of ΔΨm. Furthermore, photolysis of caged NO at levels that had no effect alone promoted a profound mitochondrial depolarisation when combined with high [Ca2+]c, either in response to KCl or to glutamate in cultures at 7–10 DIV.In cells that showed only modest mitochondrial responses to glutamate, induction of a mitochondrial depolarisation by the addition of NO was followed by a secondary rise in [Ca2+]c. These data suggest that [Ca2+]c and nitric oxide act synergistically to cause mitochondrial dysfunction and impaired [Ca2+]c homeostasis during glutamate toxicity

Glutamate-induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones

The objective of this study was to clarify the relationships between loss of mitochondrial potential and the perturbation of neuronal Ca2+ homeostasis induced by a toxic glutamate challenge. Digital fluorescence imaging techniques were employed to monitor simultaneously changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and mitochondrial potential (ΔΨm) in individual hippocampal neurones in culture coloaded with fura-2 AM or fura-2FF AM and rhodamine 123 (Rh 123).In most cells (96 %) at 6-7 days in vitro (DIV) and in a small proportion of cells (29 %) at 11-17 DIV the [Ca2+]i increase induced by exposure to 100 μm glutamate for 10 min was associated with a small mitochondrial depolarisation, followed by mitochondrial repolarisation, and a degree of recovery of [Ca2+]i following glutamate washout. In the majority of neurones at 11-17 DIV (71 %), exposure to glutamate for 10 min induced a profound mono- or biphasic mitochondrial depolarisation, which was clearly correlated with a sustained [Ca2+]i plateau despite the removal of glutamate.Addition of glutamate receptor antagonists (15 μm MK-801 plus 75 μm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)) to the washout solution did not affect the post-glutamate [Ca2+]i plateau in neurones exhibiting a profound mitochondrial depolarisation but greatly improved [Ca2+]i recovery in those neurones undergoing only a small mitochondrial depolarisation, suggesting that the release of endogenous glutamate delays [Ca2+]i recovery in the postglutamate period.Cyclosporin A (500 nM) or N-methyl Val-4-cyclosporin A (200 nM) delayed or even prevented the development of the second phase of mitochondrial depolarisation in cells at 11-17 DIV and increased the proportion of neurones exhibiting a small monophasic mitochondrial depolarisation and [Ca2+]i recovery upon glutamate removal.We have thus described a striking correlation between mitochondrial depolarisation and the failure of cells to restore [Ca2+]i following a toxic glutamate challenge. These data suggest that mitochondrial dysfunction plays a major role in the deregulation of [Ca2+]i associated with glutamate toxicity

CA2+ IMAGING PRINCIPLES OF ANALYSIS AND ENHANCEMENT

In this chapter, we review the theoretical and experimental foundations underling a quantitative approach to Ca2+ imaging, discuss equilibrium conditions and their violations and present a computational framework that can be used to estimate the spatial and temporal dynamics of Ca2+ signals based of fluorescence measurements with Ca2+ indicators