Alan Fine

CD31− but Not CD31+ Cardiac Side Population Cells Exhibit Functional Cardiomyogenic Differentiation

Heart failure remains a leading cause of morbidity and mortality. The cellular mechanism underlying the development of cardiac dysfunction is a decrease in the number of viable cardiomyocytes. Recent observations have suggested that the adult heart may contain a progenitor cell population. Side population (SP) cells, characterized by a distinct Hoechst dye efflux pattern, have been shown to exist in multiple tissues and are capable of tissue-specific differentiation. In this report, we confirm the existence of a cardiac SP cell population, immunophenotypically distinct from bone marrow SP cells. Moreover, we demonstrate that among cardiac SP cells, the greatest potential for cardiomyogenic differentiation is restricted to cells negative for CD31 expression and positive for stem cell antigen 1 (Sca1) expression (CD31−/Sca1+). Furthermore, we determine that CD31−/Sca1+ cardiac SP cells are capable of both biochemical and functional cardiomyogenic differentiation into mature cardiomyocytes, with expression of cardiomyocyte-specific transcription factors and contractile proteins, as well as stimulated cellular contraction and intracellular calcium transients indistinguishable from adult cardiomyocytes. We also determine the necessity of cell-extrinsic signaling through coupling, although not fusion, with adult cardiomyocytes in regulating cardiomyogenic differentiation of cardiac SP cells. We, therefore, conclude that CD31−/Sca1+ cardiac SP cells represent a distinct cardiac progenitor cell population, capable of cardiomyogenic differentiation into mature cardiomyocytes through a process mediated by cellular coupling with adult cardiomyocytes.

Calcium Stores in Hippocampal Synaptic Boutons Mediate Short-Term Plasticity, Store-Operated Ca2+ Entry, and Spontaneous Transmitter Release

Evoked transmitter release depends upon calcium influx into synaptic boutons, but mechanisms regulating bouton calcium levels and spontaneous transmitter release are obscure. To understand these processes better, we monitored calcium transients in axons and presynaptic terminals of pyramidal neurons in hippocampal slice cultures. Action potentials reliably evoke calcium transients in axons and boutons. Calcium-induced calcium release (CICR) from internal stores contributes to the transients in boutons and to paired-pulse facilitation of EPSPs. Store depletion activates store-operated calcium channels, influencing the frequency of spontaneous transmitter release. Boutons display spontaneous Ca2+ transients; blocking CICR reduces the frequency of these transients and of spontaneous miniature synaptic events. Thus, spontaneous transmitter release is largely calcium mediated, driven by Ca2+ release from internal stores. Bouton store release is important for short-term synaptic plasticity and may also contribute to long-term plasticity.

Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines

We have used confocal microscopy to monitor synaptically evoked Ca2+ transients in the dendritic spines of hippocampal pyramidal cells. Individual spines respond to single afferent stimuli (<0.1 Hz) with Ca2+ transients or failures, reflecting the probability of transmitter release at the activated synapse. Both AMPA and NMDA glutamate receptor antagonists block the synaptically evoked Ca2+ transients; the block by AMPA antagonists is relieved by low Mg2+. The Ca2+ transients are mainly due to the release of calcium from internal stores, since they are abolished by antagonists of calcium-induced calcium release (CICR); CICR antagonists, however, do not depress spine Ca2+ transients generated by backpropagating action potentials. These results have implications for synaptic plasticity, since they show that synaptic stimulation can activate NMDA receptors, evoking substantial Ca2+ release from the internal stores in spines without inducing long-term potentiation (LTP) or depression (LTD).

Restoration of Cardiac Progenitor Cells After Myocardial Infarction by Self-Proliferation and Selective Homing of Bone Marrow–Derived Stem Cells

Tissue-specific progenitor cells contribute to local cellular regeneration and maintain organ function. Recently, we have determined that cardiac side-population (CSP) cells represent a distinct cardiac progenitor cell population, capable of in vitro differentiation into functional cardiomyocytes. The response of endogenous CSP to myocardial injury, however, and the cellular mechanisms that maintain this cardiac progenitor cell pool in vivo remain unknown. In this report we demonstrate that local progenitor cell proliferation maintains CSP under physiologic conditions, with little contribution from extracardiac stem cell sources. Following myocardial infarction in adult mice, however, CSP cells are acutely depleted, both within the infarct and noninfarct areas. CSP pools are subsequently reconstituted to baseline levels within 7 days after myocardial infarction, through both proliferation of resident CSP cells, as well as through homing of bone marrow–derived stem cells (BMC) to specific areas of myocardial injury and immunophenotypic conversion of BMC to adopt a CSP phenotype. We, therefore, conclude that following myocardial injury, cardiac progenitor cell populations are acutely depleted and are reconstituted to normal levels by both self-proliferation and selective homing of BMC. Understanding and enhancing such processes hold enormous potential for therapeutic myocardial regeneration.

Extracellular Ca2+ Depletion Contributes to Fast Activity-Dependent Modulation of Synaptic Transmission in the Brain

Synaptic activation is associated with rapid changes in intracellular Ca(2+), while the extracellular Ca(2+) level is generally assumed to be constant. Here, using a novel optical method to measure changes in extracellular Ca(2+) at high spatial and temporal resolution, we find that brief trains of synaptic transmission in hippocampal area CA1 induce transient depletion of extracellular Ca(2+). We show that this depletion, which depends on postsynaptic NMDA receptor activation, decreases the Ca(2+) available to enter individual presynaptic boutons of CA3 pyramidal cells. This in turn reduces the probability of consecutive synaptic releases at CA3-CA1 synapses and therefore contributes to short-term paired-pulse depression of minimal responses. This activity-dependent depletion of extracellular Ca(2+) represents a novel form of fast retrograde synaptic signaling that can modulate glutamatergic information transfer in the brain.

Akt Signaling Regulates Side Population Cell Phenotype via Bcrp1 Translocation

Akt is an important regulator of cell survival, growth, and glucose metabolism in many cell types, but the role of this signaling molecule in hematopoietic stem cells is poorly defined. Side population (SP) cells are enriched for hematopoietic stem cell activity and are defined by their ability to efficiently efflux Hoechst 33342. Bone marrow from Akt1-null mice exhibited a reduced SP fraction. However, bone marrow cellularity, growth factor-responsive progenitor cultures, and engraftable stem cells were normal in these mice. Treatment of bone marrow with LY294002, an inhibitor of the Akt effector protein phosphatidylinositol 3-kinase, led to a reversible loss of the SP fraction. Bcrp1, which encodes the Hoechst dye transporter, was translocated from the membrane to the intracellular compartment under conditions that promote the SP-depleted state. Lentivirus-mediated overexpression of Akt1 in bone marrow markedly increased the SP fraction, whereas there was no effect on bone marrow from Bcrp-/- mice. These data suggest that Akt signaling modulates the SP cell phenotype by regulating the expression of Bcrp1.

NR2B-Containing Receptors Mediate Cross Talk among Hippocampal Synapses

Under some conditions, synaptically released glutamate can exert long-range actions in the cortical microcircuitry. To what extent glutamate spillover leads to direct cross talk among individual synapses remains unclear. We recorded NMDAR-mediated EPSCs in acute hippocampal slices at 35°C by stimulating two independent pathways that converge on the same CA1 pyramidal cell. Activation of a conditioning pathway in the presence of the use-dependent blocker dizocilpine maleate (MK801) resulted in partial NMDA receptor (NMDAR) blockade in the other, silent pathway. This was accompanied by an increase in the rise time of the EPSCs in the conditioning (although not the silent) pathway, implying an increase in diffusional distance from release site to NMDARs. We estimated that up to ∼30% of NMDARs contributing to EPSCs were activated by glutamate released from multiple synaptic sources; however, NMDAR-mediated synaptic cross talk was undetectable when NR2B subunit-containing receptors were blocked (but could be rescued by blocking glutamate uptake). We propose that NR2B-containing NMDARs can detect glutamate arising from multiple synapses, whereas NR2A-containing NMDARs only normally mediate direct synaptic transmission. These NMDAR isoforms thus play complementary roles in sensing global and local glutamate signals, respectively.

The Prolonged Life-Span of Alveolar Macrophages

To further examine the half-life of alveolar macrophages, chimeric CD 45.2 mice were generated through bone marrow transplantation of donor CD 45.1 cells. Before administration of donor cells, recipient mice were divided into two cohorts: the first cohort received total body irradiation; the second cohort also received irradiation-however, the thorax, head, and upper extremities were shielded with lead. Flow cytometric analysis was then performed on blood, peritoneal, and bronchoalveolar lavage cells over time to quantify engraftment. The data generated for the unshielded cohort of mice revealed a macrophage half-life of 30 days. In the shielded cohort, however, we found that by 8 months there was negligible replacement of recipient alveolar macrophages by donor cells, despite reconstitution of the blood and peritoneum by donor bone marrow. Consistent with these findings, the mean fluorescent intensity of alveolar macrophages remained stable over a 4-week period after in vivo PKH26 dye loading. Together, these data show that previous alveolar macrophage half-life studies were confounded by the fact that they did not account for the toxic effects of irradiation conditioning regimens, and demonstrate that the bone marrow does not significantly contribute to the alveolar macrophage compartment during steady-state conditions.

Learning impairments following injection of a selective cholinergic immunotoxin, ME20.4 IgG-saporin, into the basal nucleus of Meynert in monkeys

Four groups of monkeys (Callithrix jacchus) were injected with saline or increasing amounts of the immunotoxin, ME20.4 IgG-saporin, directly into the basal nucleus of Meynert via a frontal trajectory which avoided damage to the overlying basal ganglia. ME20.4 IgG binds to the primate p75 low-affinity neurotrophin receptor, when the saporin derivitized antibody is injected into the basal forebrain, it selectively destroys the magnocellular neurons of the basal nucleus of Meynert which are the cells of origin of the cholinergic projection to the neocortex. The highest dose of ME20.4 IgG-saporin produced a significant impairment on acquisition of a perceptually difficult visual discrimination. There was no significant effect on retention of tasks learnt before or after surgery, nor on concurrent acquisition of several perceptually easy discriminations or serial reversal of an easy discrimination. These results suggest that the impairment is not due to visual, motor or motivational difficulties and does not consist of difficulties with the formation of reward associations. Rather the impairment is largely confined to acquisition of perceptual discriminations. There was a significant correlation between the density of ME20.4 immunostaining in the basal nucleus of Meynert and the density of acetylcholinesterase histochemical staining in the frontal and temporal cortex and an inverse correlation between both of these and the degree of learning impairment in the animals. Lesioned animals also showed significant impairment on acquisition and reversal of perceptually easy discriminations when treated with a dose of scopolamine which did not impair performance in control animals. These results provide further evidence that cortical cholinergic neurotransmission contributes to certain forms of learning. The availability of a selective cholinergic immunotoxin effective in primates provides an important new tool for the study of cholinergic function and its involvement in ageing, Alzheimers disease and other pathological states.

Optical Quantal Analysis Reveals a Presynaptic Component of LTP at Hippocampal Schaffer-Associational Synapses

The mechanisms by which long-term potentiation (LTP) is expressed are controversial, with evidence for both presynaptic and postsynaptic involvement. We have used confocal microscopy and Ca(2+)-sensitive dyes to study LTP at individual visualized synapses. Synaptically evoked Ca(2+) transients were imaged in distal dendritic spines of pyramidal cells in cultured hippocampal slices, before and after the induction of LTP. At most synapses, from as early as 10 min to at least 60 min after induction, LTP was associated with an increase in the probability of a single stimulus evoking a postsynaptic Ca(2+) response. These observations provide compelling evidence of a presynaptic component to the expression of early LTP at Schaffer-associational synapses. In most cases, the store-dependent evoked Ca(2+) transient in the spine was also increased after induction, a novel postsynaptic aspect of LTP.