S. Brian Andrews

Calcium-dependent mitochondrial function and dysfunction in neurons

Calcium is an extraordinarily versatile signaling ion, encoding cellular responses to a wide variety of external stimuli. In neurons, mitochondria can accumulate enormous amounts of calcium, with the consequence that mitochondrial calcium uptake, sequestration and release play pivotal roles in orchestrating calcium‐dependent responses as diverse as gene transcription and cell death. In this review, we consider the basic chemistry of calcium as a ‘sticky’ cation, which leads to extremely high bound/free ratios, and discuss areas of current interest or controversy. Topics addressed include methodologies for measuring local intracellular calcium, mitochondrial calcium buffering and loading capacity, mitochondrially directed spatial calcium gradients, and the role of calcium overload‐dependent mitochondrial dysfunction in glutamate‐evoked excitotoxic injury and neurodegeneration. Finally, we consider the relationship between delayed calcium de‐regulation, the mitochondrial permeability transition and the generation of reactive oxygen species, and propose a unified view of the ‘source specificity’ and ‘calcium overload’ models of N‐methyl‐d‐aspartate (NMDA) receptor‐dependent excitotoxicity. Non‐NMDA receptor mechanisms of excitotoxicity are discussed briefly.

Co-localization of the myelin-associated glycoprotein and the microfilament components, F-actin and spectrin, in Schwann cells of myelinated nerve fibres

SummaryThe myelin-associated glycoprotein (MAG) is an intrinsic membrane protein that is specific for myelinating cells. MAG has been proposed to function in the PNS as an adhesion molecule involved in Schwann cell-axon contact and maintenance of cytoplasmic channels within the myelin sheath. In this report we show that the microfilament components, F-actin and spectrin, co-localize with MAG in periaxonal membranes, Schmidt-Lanterman incisures, paranodal myelin loops, and inner and outer mesaxons of myelinating Schwann cells. F-actin was localized light microscopically by rhodamine-labelled phallicidin binding. Spectrin and MAG were localized by light microscopic and ultrastructural immunocytochemistry. The findings indicate that plasma membrane linkage of F-actin in Schwann cells is likely to occur via spectrin, and raise the possibility that microfilaments interact with the cytoplasmic domain of MAG. An interaction between MAG and microfilaments would be consistent with the proposed function of MAG as an adhesion molecule.

Coupling diverse routes of calcium entry to mitochondrial dysfunction and glutamate excitotoxicity

Overactivation of NMDA receptors (NMDARs) is a critical early step in glutamate-evoked excitotoxic injury of CNS neurons. Distinct NMDAR-coupled pathways specified by, for example, receptor location or subunit composition seem to govern glutamate-induced excitotoxic death, but there is much uncertainty concerning the underlying mechanisms of pathway selection. Here we ask whether, and if so how, route-specific vulnerability is coupled to Ca2+ overload and mitochondrial dysfunction, which is also a known, central component of exitotoxic injury. In cultured hippocampal neurons, overactivation of only extrasynaptic NMDARs resulted in Ca2+ entry strong enough to promote Ca2+ overload, which subsequently leads to mitochondrial dysfunction and cell death. Receptor composition per se appears not to be a primary factor for specifying signal coupling, as NR2B inhibition abolished Ca2+ loading and was protective only in predominantly NR2B-expressing young neurons. In older neurons expressing comparable levels of NR2A- and NR2B-containing NMDARs, amelioration of Ca2+ overload required the inhibition of extrasynaptic receptors containing both NR2 subunits. Prosurvival synaptic stimuli also evoked Ca2+ entry through both N2A- and NR2B-containing NMDARs, but, in contrast to excitotoxic activation of extrasynaptic NMDARs, produced only low-amplitude cytoplasmic Ca2+ spikes and modest, nondamaging mitochondrial Ca2+ accumulation. The results—showing that the various routes of excitotoxic Ca2+ entry converge on a common pathway involving Ca2+ overload-induced mitochondrial dysfunction—reconcile and unify many aspects of the “route-specific” and “calcium load-dependent” views of exitotoxic injury.

Excitotoxic Calcium Overload in a Subpopulation of Mitochondria Triggers Delayed Death in Hippocampal Neurons

In neurons, excitotoxic stimulation induces mitochondrial calcium overload and the release of pro-apoptotic proteins, which triggers delayed cell death. The precise mechanisms of apoptogen release, however, remain controversial. To characterize the linkage between mitochondrial calcium load and cell vulnerability, and to test the hypothesis that only a subpopulation of mitochondria damaged by calcium overload releases apoptogens, we have measured directly the concentrations of total Ca (free plus bound) in individual mitochondria and monitored in parallel structural changes and the subcellular localization of pro-apoptotic cytochrome c after NMDA overstimulation in cultured hippocampal neurons. Beyond transient elevation of cytosolic calcium and perturbation of Na+/K+ homeostasis, NMDA stimulation induced dramatic, but mainly reversible, changes in mitochondria, including strong calcium elevation, membrane potential depolarization, and variable swelling. Elevation of matrix Ca in the approximately one-third of mitochondria that were strongly swollen, as well as the absence of swelling when Ca2+ entry was abolished, indicate an essential role for Ca overload. Shortly after NMDA exposure, cytochrome c, normally localized to mitochondria, became diffusely distributed in the cytoplasm, coincident with the appearance of severely swollen mitochondria with ruptured outer membranes; under these conditions, cytochrome c was retained in intact mitochondria, implying that it was released mainly from damaged mitochondria. Consistent with the role of mitochondrial Ca overload, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone decreased Ca accumulation, prevented cytochrome c release, and was neuroprotective. These results support a mechanism in which delayed excitotoxic death involves apoptogen release from a subpopulation of calcium-overloaded mitochondria, whereas other, undamaged mitochondria maintain normal function.

Strong Calcium Entry Activates Mitochondrial Superoxide Generation, Upregulating Kinase Signaling in Hippocampal Neurons

Large increases in cytosolic free Ca2+ ([Ca2+]i) activate several kinases that are important for neuronal plasticity, including Ca2+/calmodulin-dependent kinase II (CaMKII), protein kinase A (PKA), and protein kinase C (PKC). Because it is also known, mainly in non-neuronal systems, that superoxide radicals (O2-) activate these (and other) kinases and because O2- generation by mitochondria is in part [Ca2+]i dependent, we examined in hippocampal neurons the relationship between Ca2+ entry, O2- production, and kinase activity. We found that, after large stimulus-induced [Ca2+]i increases, O2- selectively produced by mitochondria near plasmalemmal sites of Ca2+ entry acts as a modulator to upregulate the two kinases, namely, CaMKII and PKA, whose activities are directly or indirectly phosphorylation dependent. The common mechanism involves O2- inhibition of inactivating protein phosphatases. Conversely, because small [Ca2+]i increases do not promote mitochondrial respiration and O2- generation, weak stimuli favor enhanced phosphatase activity, which therefore leads to suppressed kinase activity. Enhanced O2- production also promoted PKC activity but by a phosphatase-independent pathway. These results suggest that Ca2+-dependent upregulation of mitochondrial O2- production may be a general mechanism for linking Ca2+ entry to enhanced kinase activity and therefore to synaptic plasticity. This mechanism also represents yet another way that mitochondria, acting as calcium sensors, can play a role in neuronal signal transduction.

The Bcl-2 Gene Polymorphism rs956572AA Increases Inositol 1,4,5-Trisphosphate Receptor–Mediated Endoplasmic Reticulum Calcium Release in Subjects with Bipolar Disorder

BACKGROUND Bipolar disorder (BPD) is characterized by altered intracellular calcium (Ca(2+)) homeostasis. Underlying mechanisms involve dysfunctions in endoplasmic reticulum (ER) and mitochondrial Ca(2+) handling, potentially mediated by B-cell lymphoma 2 (Bcl-2), a key protein that regulates Ca(2+) signaling by interacting directly with these organelles, and which has been implicated in the pathophysiology of BPD. Here, we examined the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular Ca(2+) dynamics in patients with BPD. METHODS Live cell fluorescence imaging and electron probe microanalysis were used to measure intracellular and intra-organelle free and total calcium in lymphoblasts from 18 subjects with BPD carrying the AA, AG, or GG variants of the rs956572 SNP. Analyses were carried out under basal conditions and in the presence of agents that affect Ca(2+) dynamics. RESULTS Compared with GG homozygotes, variant AA-which expresses significantly reduced Bcl-2 messenger RNA and protein-exhibited elevated basal cytosolic Ca(2+) and larger increases in inositol 1,4,5-trisphosphate receptor-mediated cytosolic Ca(2+) elevations, the latter in parallel with enhanced depletion of the ER Ca(2+) pool. The aberrant behavior of AA cells was reversed by chronic lithium treatment and mimicked in variant GG by a Bcl-2 inhibitor. In contrast, no differences between SNP variants were found in ER or mitochondrial total Ca(2+) content or in basal store-operated Ca(2+) entry. CONCLUSIONS These results demonstrate that, in patients with BPD, abnormal Bcl-2 gene expression in the AA variant contributes to dysfunctional Ca(2+) homeostasis through a specific ER inositol 1,4,5-trisphosphate receptor-dependent mechanism.

Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro.

We studied the development of transverse (T)-tubules and sarcoplasmic reticulum (SR) in relationship to myofibrillogenesis in normal and dysgenic (mdg/mdg) mouse skeletal muscle by immunofluorescent labeling of specific membrane and myofibrillar proteins. At E16 the development of the myofibrils and membranes in dysgenic and normal diaphragm was indistinguishable, including well developed myofibrils, a delicate network of T-tubules, and a prominent SR which was not yet cross-striated. In diaphragms of E18 dysgenic mice, both the number and size of muscle fibers and myofibrillar organization were deficient in comparison to normal diaphragms, as previously reported. T-tubule labeling was abnormal, showing only scattered tubules and fragments. However, many muscle fibers displayed cross striation of sarcomeric proteins and SR comparable to normal muscle. In cultured myotubes, cross-striated organization of sarcomeric proteins proceeded essentially in two stages: first around the Z-line and later in the A-band. Sarcomeric organization of the SR coincided with the first stage, while the appearance of T-tubules in the mature transverse orientation occurred infrequently, only after A-band maturation. In culture, myofibrillar and membrane organization was equivalent in normal and dysgenic muscle at the earlier stage of development, but half as many dysgenic myotubes reached the later stage as compared to normal. We conclude that the mdg mutation has little effect on the initial stage of membrane and myofibril development and that the deficiencies often seen at later stages result indirectly from the previously described absence of dihydropyridine receptor function in the mutant.

Real-time measurement of free Ca2+ changes in CNS myelin by two-photon microscopy

Here we describe a technique for measuring changes in Ca2+ in the cytosolic domain of mature compact myelin of live axons in the central nervous system (CNS). We label the myelin sheath of optic nerve and dorsal column axons by using the Ca2+ indicator X-rhod-1 coupled with DiOC6(3) to produce bright myelin counterstaining, thereby providing unambiguous identification of the myelin sheath for analysis of two-photon excited fluorescence. We present evidence for localization of the Ca2+ reporter to the cytosolic domain of myelin, obtained by using fluorescence lifetime, spectral measurements and Mn2+ quenching. Chemical ischemia increased myelinic X-rhod-1 fluorescence (∼50% after 30 min) in a manner dependent on extracellular Ca2+. Inhibiting Na+-dependent glutamate transporters (with TBOA) or glycine transporters (with sarcosine and ALX-1393) reduced the ischemia-induced increase in Ca2+. We show that myelinic N-methyl-D-aspartate (NMDA) receptors are activated by the two conventional coagonists glutamate and glycine, which are released by specific transporters under conditions of cellular Na+ loading and depolarization in injured white matter. This new technique facilitates detailed studies of living myelin, a vital component of the mammalian CNS.

Microheterogeneity of calcium signalling in dendrites

Transient changes in the intracellular concentration of free Ca2+ ([Ca2+]i) originating from voltage‐ or ligand‐gated influx and by ligand‐ or Ca2+‐gated release from intracellular stores, trigger or modulate many fundamental neuronal processes, including neurotransmitter release and synaptic plasticity. Of the intracellular compartments involved in Ca2+ clearance, the endoplasmic reticulum (ER) has received the most attention because it expresses Ca2+ pumps and Ca2+ channels, thus endowing it with the potential to act as both an intracellular calcium sink and store. We review here our ongoing work on the role of calcium sequestration into, and release from, ER cisterns and the role that this plays in the generation and termination of free [Ca2+]i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. These studies have been approached by combining parallel microfluorometric measurements of free cytosolic [Ca2+]i transients with energy‐dispersive X‐ray microanalytical measurements of total Ca content within specific dendritic compartments at the electron microscopy level. Our observations support the emerging realization that specific subsets of dendritic ER cisterns provide spatial and temporal microheterogeneity of Ca2+ signalling, acting not only as a major intracellular Ca sink involved in active clearance mechanisms after voltage‐ and ligand‐gated Ca2+ influx, but also as an intracellular Ca2+ source that can be mobilized by a signal cascade originating at activated synapses.

Differential NMDA receptor-dependent calcium loading and mitochondrial dysfunction in CA1 vs. CA3 hippocampal neurons

Hippocampal CA1 pyramidal neurons are selectively vulnerable to ischemia, while adjacent CA3 neurons are relatively resistant. Although glutamate receptor-mediated mitochondrial Ca(2+) overload and dysfunction is a major component of ischemia-induced neuronal death, no direct relationship between selective neuronal vulnerability and mitochondrial dysfunction has been demonstrated in intact brain preparations. Here, we show that in organotypic slice cultures NMDA induces much larger Ca(2+) elevations in vulnerable CA1 neurons than in resistant CA3. Consequently, CA1 mitochondria exhibit stronger calcium accumulation, more extensive swelling and damage, stronger depolarization of their membrane potential, and a significant increase in ROS generation. NMDA-induced Ca(2+) and ROS elevations were abolished in Ca(2+)-free medium or by NMDAR antagonists, but not by zinc chelation. We conclude that Ca(2)(+) overload-dependent mitochondrial dysfunction is a determining factor in the selective vulnerability of CA1 neurons.