Jane H.-C. Lin

Uniquely hominid features of adult human astrocytes.

Defining the microanatomic differences between the human brain and that of other mammals is key to understanding its unique computational power. Although much effort has been devoted to comparative studies of neurons, astrocytes have received far less attention. We report here that protoplasmic astrocytes in human neocortex are 2.6-fold larger in diameter and extend 10-fold more GFAP (glial fibrillary acidic protein)-positive primary processes than their rodent counterparts. In cortical slices prepared from acutely resected surgical tissue, protoplasmic astrocytes propagate Ca2+ waves with a speed of 36 μm/s, approximately fourfold faster than rodent. Human astrocytes also transiently increase cystosolic Ca2+ in response to glutamatergic and purinergic receptor agonists. The human neocortex also harbors several anatomically defined subclasses of astrocytes not represented in rodents. These include a population of astrocytes that reside in layers 5–6 and extend long fibers characterized by regularly spaced varicosities. Another specialized type of astrocyte, the interlaminar astrocyte, abundantly populates the superficial cortical layers and extends long processes without varicosities to cortical layers 3 and 4. Human fibrous astrocytes resemble their rodent counterpart but are larger in diameter. Thus, human cortical astrocytes are both larger, and structurally both more complex and more diverse, than those of rodents. On this basis, we posit that this astrocytic complexity has permitted the increased functional competence of the adult human brain.

Glutamate release promotes growth of malignant gliomas

Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative diseases. Although recent data show that cultured glioma cells secrete glutamate, the growth potential of brain tumors has not yet been linked to an excitotoxic mechanism. Using bioluminescence detection of glutamate release from freshly prepared brain slices, we show that implanted glioma cells continue to secrete glutamate. Moreover, gliomas with high glutamate release have a distinct growth advantage in host brain that is not present in vitro. Treatment with the NMDA receptor antagonists MK801 or memantine slowed the growth of glutamate-secreting tumors in situ, suggesting that activation of NMDA receptors facilitates tumor expansion. These findings support a new approach for therapy of brain tumors, based upon antagonizing glutamate secretion or its target receptors.

The Transcriptome and Metabolic Gene Signature of Protoplasmic Astrocytes in the Adult Murine Cortex

Protoplasmic astrocytes are critically important to energy metabolism in the CNS. Our current understanding of the metabolic interactions between neurons and glia is based on studies using cultured cells, from which mainly inferential conclusions have been drawn as to the relative roles of neurons and glia in brain metabolism. In this study, we used functional genomics to establish the relative compartmentalization of neuronal and astrocytic metabolic pathways in the adult brain. To this end, fluorescence-activated cell sorting was used to directly isolate neurons and protoplasmic astrocytes from the cortex of adult mice. Microarray analysis showed that astrocytes and neurons each express transcripts predicting individual self-sufficiency in both glycolysis and oxidative metabolism. Surprisingly, most enzymes in the tricarboxylic acid (TCA) cycle were expressed at higher relative levels in astrocytes than in neurons. Mass spectrometric analysis of the TCA cycle intermediates confirmed that freshly isolated adult astrocytes maintained an active TCA cycle, whereas immuno-electron microscopy revealed that fine astrocytic processes encompassing synapses contained a higher density of mitochondria than surrounding cells. These observations indicate that astrocytes exhibit robust oxidative metabolism in the intact adult brain and suggest a prominent contribution of astrocytic metabolism to functional brain imaging, including BOLD (blood-oxygen level-dependent) functional magnetic resonance imaging signals.

Intercellular calcium signaling mediated by point-source burst release of ATP

Calcium signaling, manifested as intercellular waves of rising cytosolic calcium, is, in many cell types, the result of calcium-induced secretion of ATP and activation of purinergic receptors. The mechanism by which ATP is released has hitherto not been established. Here, we show by real-time bioluminescence imaging that ATP efflux is not uniform across a field of cells but is restricted to brief, abrupt point-source bursts. The ATP bursts emanate from single cells and manifest the transient opening of nonselective membrane channels, which admits fluorescent indicators of ≤1.5 kDa. These observations challenge the existence of regenerative ATP release, because ATP efflux is finite and restricted to a point source. Transient efflux of cytosolic nucleotides from a subset of cells may represent a conserved pathway for coordinating local activity of electrically nonexcitable cells, because identical patterns of ATP release were identified in human astrocytes, endothelial cells, and bronchial epithelial cells.

Astrocytic gap junctions remain open during ischemic conditions.

Gap junctions are highly conductive channels that allow the direct transfer of intracellular messengers such as Ca2+and inositol triphosphate (IP3) between interconnected cells. In brain, astrocytes are coupled extensively by gap junctions. We found here that gap junctions among astrocytes in acutely prepared brain slices as well as in culture remained open during ischemic conditions. Uncoupling first occurred after the terminal loss of plasma membrane integrity. Gap junctions therefore may link ischemic astrocytes in an evolving infarct with the surroundings. The free exchange of intracellular messengers between dying and potentially viable astrocytes might contribute to secondary expansion of ischemic lesions.

A Central Role of Connexin 43 in Hypoxic Preconditioning

Preconditioning is an endogenous mechanism in which a nonlethal exposure increases cellular resistance to subsequent additional severe injury. Here we show that connexin 43 (Cx43) plays a key role in protection afforded by preconditioning. Cx43 null mice were insensitive to hypoxic preconditioning, whereas wild-type littermate mice exhibited a significant reduction in infarct volume after occlusion of the middle cerebral artery. In cultures, Cx43-deficient cells responded to preconditioning only after exogenous expression of Cx43, and protection was attenuated by small interference RNA or by channel blockers. Our observations indicate that preconditioning reduced degradation of Cx43, resulting in a marked increase in the number of plasma membrane Cx43 hemichannels. Consequently, efflux of ATP through hemichannels led to accumulation of its catabolic product adenosine, a potent neuroprotective agent. Thus, adaptive modulation of Cx43 can offset environmental stress by adenosine-mediated elevation of cellular resistance.

Adhesive Properties of Connexin Hemichannels

Gap junctions are intercellular channels formed by hemichannels (or connexons) from two neighboring cells. Hemichannels, which are composed of proteins called connexins, can function as conduits of ATP and glutamate, and interact with adhesion molecules and other signaling elements. As a result, their functional repertoire is expanding into other roles, such as control of cell growth or cell migration. Here we further elucidate the involvement of hemichannels in cell–cell adhesion by analyzing how connexins regulate cell adhesion without the need of gap junction formation. Using a short‐term aggregation assay with C6‐glioma and HeLa cells stably transfected with connexin (Cx) 43 or Cx32, we found that the connexin type dictates the ability of these cells to aggregate, even though these two cell types do not usually adhere to each other. We have also found that high expression of Cx43, but not Cx32 hemichannels, can drive adhesion of cells expressing low levels of Cx43. Aggregation was not dependent on high levels of extracellular Ca2+, as Ca2+ removal did not change the aggregation of Cx43‐expressing cells. Our data confirm that connexin hemichannels can establish adhesive interactions without the need for functional gap junctions, and support the concept that connexins act as adhesion molecules independently of channel formation.

Expression and function of astrocytic gap junctions in aging.

Astrocytic gap junctions have been implicated in a variety of signaling pathways essential to normal brain function. However, no information exists on the prevalence of gap junction channels and their function in the aging brain. Here we have compared the expression of the two most abundant astrocytic gap junction proteins in young and senescent brains and quantified the extent of functional gap junction coupling. The expression level of Cx43 peaked in 7-month-old mice. The relative numbers of Cx43 immunoreactive plaques were 596+/-61, 734+/-62, and 755+/-114 in 3-, 7-, and 21-month-old mice, whereas plaques size averaged 0.9+/-0.1 microm(2) (3 months), 1.3+/-0.1 microm(2) (7 months), and 0.7+/-0.1 microm(2) (21 months). The expression level of Cx30 was also highest in 7-month-old animals (315+/-49 plaques, size 0.8+/-0.07 microm(2) vs. 585+/-51 plaques, size 0.9+/-0.1 microm(2) in 3- and 7-month-old mice, respectively), but only 262+/-63 plaques (size 0.4+/-0.04 microm(2)) in 21-month-old mice. Western blot analysis revealed that the content of both Cx43 and Cx30 remained relatively constant at 3, 7, and 21 months. The fluorescence recovery of photobleach technique (FRAP) was used to evaluate coupling in freshly prepared hippocampal slices. Gap junction coupling did not change significantly as a function of aging, but a tendency towards reduced coupling was observed as the animals aged. Average fluorescence recovery after 2 min was 63+/-6% in younger animals, 59+/-5% in adult animals, and 54+/-4% in old brain. These observations indicate that although astrocytic gap junction proteins are maintained at high levels through the entire lifespan of mice, aging is associated with changes in the number and size of both Cx30 and Cx43 gap junction plaques.