Maria Luisa Cotrina

Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury

Traumatic spinal cord injury is characterized by an immediate, irreversible loss of tissue at the lesion site, as well as a secondary expansion of tissue damage over time. Although secondary injury should, in principle, be preventable, no effective treatment options currently exist for patients with acute spinal cord injury (SCI). Excessive release of ATP by the traumatized tissue, followed by activation of high-affinity P2X7 receptors, has previously been implicated in secondary injury, but no clinically relevant strategy by which to antagonize P2X7 receptors has yet, to the best of our knowledge, been reported. Here we have tested the neuroprotective effects of a systemically administered P2X7R antagonist, Brilliant blue G (BBG), in a weight-drop model of thoracic SCI in rats. Administration of BBG 15 min after injury reduced spinal cord anatomic damage and improved motor recovery without evident toxicity. Moreover, BBG treatment directly reduced local activation of astrocytes and microglia, as well as neutrophil infiltration. These observations suggest that BBG not only protected spinal cord neurons from purinergic excitotoxicity, but also reduced local inflammatory responses. Importantly, BBG is a derivative of a commonly used blue food color (FD&C blue No. 1), which crosses the blood–brain barrier. Systemic administration of BBG may thus comprise a readily feasible approach by which to treat traumatic SCI in humans.

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.

Astrocytes and Ischemic Injury

Ischemic injury is traditionally viewed from an axiomatic perspective of neuronal loss. Yet the ischemic infarct encompasses all cell types, including astrocytes. This review will discuss the idea that astrocytes play a fundamental role in the pathogenesis of ischemic neuronal death. It is proposed that stroke injury is primarily a consequence of the failure of astrocytes to support the essential metabolic needs of neurons. This “gliocentric view” of stroke injury predicts that pharmacological interventions specifically targeting neurons are unlikely to succeed, because it is not feasible to preserve neuronal viability in an environment that fails to meet essential metabolic requirements. Neuroprotective efforts targeting the functional integrity of astrocytes may constitute a superior strategy for future neuroprotection.

Astrocytes in the aging brain.

Astrocytes have traditionally been viewed as passive supportive cells, which were primarily responsible for maintaining an optimal environment for electrical neuronal activity. Recent studies have, however, demonstrated that the activity of nerve cells can be modulated by astrocytes, in that neurons are recruited into astrocyte-initiated and propagated calcium waves, both in vitro and in situ. By this means, propagated shifts in cytosolic calcium within the astrocytic syncytium may regulate neuronal response and firing thresholds. In turn, astrocytes are actively modulated by neuronal activity, and the existence of astrocyte–neuron signaling loops has been established in several areas of the brain. As a result of these findings, it is now recognized that astrocytes play an active role in brain function, particularly within the highly coupled astrocytic syncytium of the neocortex and the hippocampus. The mechanisms by which calcium signaling is propagated and how it is evoked are the focus of intense research activity. It is known that gap junctions and the connexins, their constituent proteins, together with the local cytoskeleton, the calcium buffer capacity, and calcium waves triggered by purinergic transmitters, all cooperate to modulate astrocytic signaling to neighboring cells in young animals. What changes do astrocytes and their signaling machinery undergo during the aging process? This is a question of paramount importance; altered astrocytic dynamics in the aged brain may alter synaptic efficacy and neuronal survival and perhaps contribute to the cognitive decline observed during aging. In this review, we analyze our current understanding of astrocytic function during aging by reexamining the mechanisms by which astrocytes contribute to neuronal function and survival in normal brain and the changes they undergo in the aged brain.

Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering

Ammonia is a ubiquitous waste product of protein metabolism that can accumulate in numerous metabolic disorders, causing neurological dysfunction ranging from cognitive impairment to tremor, ataxia, seizures, coma and death. The brain is especially vulnerable to ammonia as it readily crosses the blood-brain barrier in its gaseous form, NH3, and rapidly saturates its principal removal pathway located in astrocytes. Thus, we wanted to determine how astrocytes contribute to the initial deterioration of neurological functions characteristic of hyperammonemia in vivo. Using a combination of two-photon imaging and electrophysiology in awake head-restrained mice, we show that ammonia rapidly compromises astrocyte potassium buffering, increasing extracellular potassium concentration and overactivating the Na+-K+-2Cl− cotransporter isoform 1 (NKCC1) in neurons. The consequent depolarization of the neuronal GABA reversal potential (EGABA) selectively impairs cortical inhibitory networks. Genetic deletion of NKCC1 or inhibition of it with the clinically used diuretic bumetanide potently suppresses ammonia-induced neurological dysfunction. We did not observe astrocyte swelling or brain edema in the acute phase, calling into question current concepts regarding the neurotoxic effects of ammonia. Instead, our findings identify failure of potassium buffering in astrocytes as a crucial mechanism in ammonia neurotoxicity and demonstrate the therapeutic potential of blocking this pathway by inhibiting NKCC1.

Contributions of the Na+/K+-ATPase, NKCC1, and Kir4.1 to hippocampal K+ clearance and volume responses

Network activity in the brain is associated with a transient increase in extracellular K+ concentration. The excess K+ is removed from the extracellular space by mechanisms proposed to involve Kir4.1‐mediated spatial buffering, the Na+/K+/2Cl− cotransporter 1 (NKCC1), and/or Na+/K+‐ATPase activity. Their individual contribution to [K+]o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na+/K+‐ATPase and to resolve their involvement in clearance of extracellular K+ transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K+]o increases above basal levels. Increased [K+]o produced NKCC1‐mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K+ clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K+ removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K+]o increase. In contrast, inhibition of the different isoforms of Na+/K+‐ATPase reduced post‐stimulus clearance of K+ transients. The astrocyte‐characteristic α2β2 subunit composition of Na+/K+‐ATPase, when expressed in Xenopus oocytes, displayed a K+ affinity and voltage‐sensitivity that would render this subunit composition specifically geared for controlling [K+]o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na+/K+‐ATPase accounted for the stimulus‐induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity‐induced extracellular K+ recovery in native hippocampal tissue while Kir4.1 and Na+/K+‐ATPase serve temporally distinct roles. GLIA 2014;62:608–622

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.

Physiological and pathological functions of P2X7 receptor in the spinal cord

ATP-mediated signaling has widespread actions in the nervous system from neurotransmission to regulation of proliferation. In addition, ATP is released during injury and associated to immune and inflammatory responses. Still, the potential of therapeutic intervention of purinergic signaling during pathological states is only now beginning to be explored because of the large number of purinergic receptors subtypes involved, the complex and often overlapping pharmacology and because ATP has effects on every major cell type present in the CNS. In this review, we will focus on a subclass of purinergic-ligand-gated ion channels, the P2X7 receptor, its pattern of expression and its function in the spinal cord where it is abundantly expressed. We will discuss the mechanisms for P2X7R actions and the potential that manipulating the P2X7R signaling pathway may have for therapeutic intervention in pathological events, specifically in the spinal cord.

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.

Regulation of EphB2 activation and cell repulsion by feedback control of the MAPK pathway

In this study, we investigated whether the ability of Eph receptor signaling to mediate cell repulsion is antagonized by fibroblast growth factor receptor (FGFR) activation that can promote cell invasion. We find that activation of FGFR1 in EphB2-expressing cells prevents segregation, repulsion, and collapse responses to ephrinB1 ligand. FGFR1 activation leads to increased phosphorylation of unstimulated EphB2, which we show is caused by down-regulation of the leukocyte common antigen–related tyrosine phosphatase receptor that dephosphorylates EphB2. In addition, FGFR1 signaling inhibits further phosphorylation of EphB2 upon stimulation with ephrinB1, and we show that this involves a requirement for the mitogen-activated protein kinase (MAPK) pathway. In the absence of activated FGFR1, EphB2 activates the MAPK pathway, which in turn promotes EphB2 activation in a positive feedback loop. However, after FGFR1 activation, the induction of Sprouty genes inhibits the MAPK pathway downstream of EphB2 and decreases cell repulsion and segregation. These findings reveal a novel feedback loop that promotes EphB2 activation and cell repulsion that is blocked by transcriptional targets of FGFR1.