60. TISSUE PLASMINOGEN ACTIVATOR SIGNALING IN THE NORMAL AND DISEASED BRAIN
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60 Tissue Plasminogen Activator Signaling in the Normal and Diseased Brain R. Bronstein, S.E. Tsirka Stony Brook University, Stony Brook, NY, United States
Tissue-type plasminogen activator (tPA) is currently the only U.S. Food and Drug Administration–approved therapy for the acute treatment of ischemic stroke . The most extensively studied function of tPA is its primary activity, namely, the proteolytic conversion of the zymogen plasminogen (plg) into the active protease plasmin, which in turn is essential for the lysis of blood clots . Human tPA is a serine protease composed of 527 residues with four functional domains on its A chain [finger, epidermal growth factor (EGF), kringle] and one (protease) on the B chain . In the central nervous system (CNS) tPA is expressed in neurons and glial cells and released in an activity-dependent manner via exocytosis . Its activity is regulated through specific protein inhibitors, plasminogen activator inhibitor 1, and neuroseprin. It does not have one specific receptor, but can function on and modulate other receptors and components of the extracellular matrix (ECM). In addition to serving as a critical hemolytic node during the fibrinolysis cascade, tPA is involved in a number of other important functions in the brain and spinal cord. In the brain, these divergent roles broadly impact the normal as well as ischemic cerebral vasculature and parenchymal structures. The preponderance of this primer will focus on the critical role that neuronal and glial tPA signaling in the normal brain, and how this signaling is perturbed in the ischemic cerebrum.
SIGNALING IN THE NORMAL CNS Signaling Through N-Methyl-d-Aspartate Receptors A role for tPA in either normal synaptic function (neuroprotection) or exaggerated neuronal stimulation
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(excitotoxicity) through glutamate receptors has been an area of persistent investigation . Endogenously, tPA expressed in hippocampal neurons is synthesized in the synaptodendritic compartment and is rapidly upregulated upon metabotropic glutamate receptor activation in a mechanism that involves regulated cytoplasmic polyadenylation . N-methyl-d-aspartate receptors (NMDARs) are members of a large family of ionotropic glutamate receptors, which mediate fast synaptic transmission in the CNS. These receptors are obligatory heterotetramers made up of eight alternatively spliced isoforms (GluN1, GluN2, and/or GluN3), with the first and third binding glycine and second binding l-glutamate. They are essential components of the synaptic cleft, involved in calciummediated glutamatergic neurotransmission essential for processes as diverse as movement and memory consolidation. They can also be found extrasynaptically, a localization thought to primarily underpin their function in excitotoxic neuronal death . Recombinant tPA administration to cultured hippocampal neurons affects calcium flux. Following stimulation of glutamate release presynaptically, recombinant tPA was reported to inhibit the resultant synchronous spontaneous calcium oscillations . The proteolytic activity of tPA was critical for NMDAR-mediated calcium currents, as the enzymatically inactive tPA mutant, with the active site Ser478 residue mutated to alanine, no longer had any effect . To control for active plasmin being the causal agent of changes in calcium flux, cultures were further incubated with α2-antiplasmin, which ablated tPA-mediated calcium signaling . In the totality of the literature, the interactions between NMDARs and tPA have been most heavily scrutinized given the explicit need to understand physiological signaling, which is eventually perturbed in various disease states including ischemia.
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Signaling in the Diseased CNS
Signaling Through Proteolytic Cleavage of ECM tPA is secreted in a single-chain form (sc-tPA) from neurons and glial cells and processed into a two-chain form (tc-tPA) by plasmin or kallikreins. Proteolytic activation of tPA does not preclude sc-tPA from being proteolytically active, as the single-chain form can act as an effector of epidermal growth factor receptor (EGFR) and N-methyl-d-aspartate (NMDA) signaling. Both sc-tPA and tc-tPA modulate the cross talk between EGFR and NMDA receptors on neurons: tPA-mediated EGFR activation leads to a downregulation of NMDAR function. Low levels of sc-tPA and tc-tPA are thought to mediate this cross talk, which clearly points to an antiexcitotoxic effect of tPA on neurons, as opposed to that seen with high-level administration of tPA. Another pointed role for tPA involves its effect on neurite outgrowth either during development and/or neurogenesis, or following injury . tPA has been shown to degrade ECM components through the generation of plasmin, allowing the extension of neurites under normal developmental cues or following temporal lobe epilepsy (TLE) . Some of these pathways have implicated phospholipase-D1 (Pld1) as the driver of tPA secretion from the growth cone in an excitation-dependent manner, thus regulating the neurite outgrowth necessary during normal hippocampal development, as well as aberrant growth due to excitotoxic insult . The mechanism of how Pld1 might promote vesicular tPA release is unclear, likely involving the activation of protein kinase C (PKC) through generation of phosphatidic acid, and possibly diacylglycerides, both of which can stimulate PKC at the cell membrane . These results clearly outline a role for tPA exocytosis from neurons in the normal growth and sprouting of neurites as well as that potentially caused by TLE.
Other Interactions Outside of its well-established interaction with NMDARs, tPA maintains other prominent roles in the CNS. In addition to the roles outlined earlier, which mostly focus on the adult animal, tPA has been shown to be an important player during development. The growth and organization of the cerebellar nuclei are critical for innate and learned motor behavior, and several groups have described a role for tPA in this process . The primary output of the cerebellar cortex comes from the actions of Purkinje neurons (PNs), sculpted by recurrent inhibitory networks within the cerebellum . As mentioned previously, PKC and its several isoforms that are expressed in the CNS are thought to be stimulated by the local levels and activity of tPA [6,7]. PKCγ activity rather than its levels are reported to be modulated by endogenous or exogenous tPA leading to the suppression of PN dendritic development .
The other predominant cell type within the cerebellum, responsible for modulating the excitatory tone onto the PNs is the granule cell, employing the parallel fibers (PFs), which synapse on the dendritic spines of PNs . The structure of these very important PF–PN synapses is thought to be structurally altered by brain-derived neurotrophic factor (BDNF) levels. BDNF production is tightly controlled by tPA within the granule cells of the cerebellum . A causal role for tPA in cerebellar development and the pruning of immature PNs has yet to be established; however, there is increasing evidence that endogenous levels of tPA play an important role through signaling effectors in this system . Mature BDNF levels are increased by the proteolytic action of tPA to plasmin, which cleaves proBDNF to BDNF, an increase abrogated by the administration of NMDAR inhibitor MK801, which is then associated with TrkB activation.
SIGNALING IN THE DISEASED CNS Signaling Through NMDA Receptors Excitotoxic neuronal injury overloads the neurons and drives them to apoptotic death . A role for tPA in NMDR function has long been postulated starting with a 2001 study indicating that NMDAR-linked excitotoxic cell death is enhanced by the presence of ∼280 nM of tPA acting through the proteolytic cleavage of the NMDA NR1 subunit in a plasminogen-independent fashion . Cerebral ischemia is a prime culprit in the generation of large excitotoxic zones, and therefore the NR1 subunit has been considered as a potential therapeutic target in the treatment of stroke and other brain pathologies . More recent in vivo findings, however, demonstrated that at the more realistic, low concentrations found in the brain, tPA actually protects neurons against excitotoxin-induced cell death . Additional totally divergent roles for the interaction between tPA and NMDARs have been proposed. For example, one group demonstrated that the interaction between tPA and NMDARs is critical in neurovascular coupling, a process by which the activation of NMDARs through neuronal activity leads to the release of nitric oxide, an essential vasodilator in the CNS . These results demonstrate that it is potentially tPA binding to different sites and/or subunit configurations of the NMDAR complex that underpins its specific role in the healthy or diseased brain.
Signaling Through EGF Receptors Most studies of the contribution of tPA to excitotoxicity have focused on extracellular tPA at the behest of cytosolic tPA. Intracellular tPA may serve as a stand-alone signaling substrate and, although tPA has been shown to
IV. MOLECULAR MECHANISMS
60. TISSUE PLASMINOGEN ACTIVATOR SIGNALING IN THE NORMAL AND DISEASED BRAIN
FIGURE 60.1 Diverse pathways engaging tissue-type plasminogen activator (tPA). tPA, acting either as a protease or nonproteolytically, can regulate neuronal activity and glial cell activation and motility. Its own expression is increased with neuronal activity both transcriptionally and posttranscriptionally within the cell, and through the action of protease inhibitors [plasminogen activator inhibitor 1 (PAI1) and neuroseprin]. BDNF, Brain-derived neurotrophic factor; ECM, extracellular matrix; LRP, lipoprotein-receptor related protein; NMDAR, N-methyl-d-aspartate receptor.
play a role in NMDAR signaling, the intracellular component has not been adequately looked at. Some evidence has emerged that EGFR plays an important role within neuronal somata. One study has demonstrated that cultured hippocampal neurons exposed to oxygen/glucose deprivation, a prominent model of ischemic injury, have varied reactions to the absence of normal endogenous levels of tPA. This effect seems to be specific to EGFRs, as perturbations of NMDARs and lipoprotein-receptor related proteins (LRPs) during titration of endogenous tPA levels did not have an effect on the level of neuroprotection. However, mitigating the interaction between endogenous tPA and EGFRs predisposed these cells to excitotoxic injury and cell death. Overall it appears that intracellular and extracellular tPAs have different substrates as well as functions, potentially contributing to excitotoxic insult outside the cell while being neuroprotective from within.
Glial Activation and tPA In addition, cytokine-like behavior of tPA has been described in the CNS. This involves the enhancement of microglial activation through nonproteolytic interactions with the heterotetramer Annexin A2/p11 complex, which result in activation of the integrin-linked kinase pathway. tPA’s interaction with LRP1, a member of the LDL receptor family, has been shown to induce the activation of matrix metalloproteinase 9 through activation of Mek1 and (extracellular-signal-regulated kinases) ERK1/2, and inflammatory nuclear factor κB signaling. Association of tPA with LRP1 was also described as a facilitator of transcytosis across the blood–brain barrier and into the CNS. tPA signaling through LRP and other effectors has also been shown to regulate macrophage and microglial
cell migration, and the activation of the Rac1 pathway . This migration has been described mainly in pathological settings, such as hemorrhagic stroke and epilepsy. The engagement of chemokines and their receptors (e.g., CCL2/CCR2) is mostly responsible for the cell motility and migration.
CONCLUSION We have put forth evidence for tPA participating in a diverse array of physiologically and pathologically relevant neural mechanisms (Fig. 60.1). Classically this critical component of the normal clotting cascade has been implicated in functions as divergent as ECM clearance through the generation of plasmin, to cytosolic sc-tPA signaling leading to activation of the EGF receptor family, or nonproteolytic, cytokine functions. These varied roles point to tPA as a highly modular, versatile protein capable of great subunit specificity when it comes to carrying out its job in the physiological and injured CNS. The multitude of roles and functions in different settings also suggests that a single approach toward its exogenous administration or its inhibition, needs to be designed very carefully, as it may result in consequences irrelevant to its intended action.
References  Yepes M, Roussel BD, Ali C, Vivien D. Tissue-type plasminogen activator in the ischemic brain: more than a thrombolytic. Trends Neurosci 2009;32:48–55.  Nolin WB, Emmetsberger J, Bukhari N, Zhang Y, Levine JM, Tsirka SE. tPA-mediated generation of plasmin is catalyzed by the proteoglycan NG2. Glia 2008;56:177–89.
IV. MOLECULAR MECHANISMS
 Park L, Gallo EF, Anrather J, Wang G, Norris EH, Paul J, et al. Key role of tissue plasminogen activator in neurovascular coupling. Proc Natl Acad Sci USA 2008;105:1073–8.  Shin CY, Kundel M, Wells DG. Rapid, activity-induced increase in tissue plasminogen activator is mediated by metabotropic glutamate receptor-dependent mRNA translation. J Neurosci 2004;24:9425–33.  Robinson SD, Lee TW, Christie DL, Birch NP. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons. Front Cell Neurosci 2015;9:404.  Zhang Y, Kanaho Y, Frohman M, Tsirka S. Regulated secretion of tissue plasminogen activator during neurite outgrowth. J Neurosci 2005;25:1797–805.
 Li J, Yu L, Gu X, Ma Y, Pasqualini R, Arap W, et al. Tissue plasminogen activator regulates Purkinje neuron development and survival. Proc Natl Acad Sci USA 2013;110:E2410–9.  Tsirka S, Gualandris A, Amaral D, Strickland S. Excitotoxin induced neuronal degeneration and seizure are mediated by tissue-type plasminogen activator. Nature 1995;377:340–4.  Yepes M. Tissue-type plasminogen activator is a neuroprotectant in the central nervous system. Front Cell Neurosci 2015;9:304.  Lin L, Hu K. Tissue plasminogen activator and inflammation: from phenotype to signaling mechanisms. Am J Clin Exp Immunol 2014;3:30–6.
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61 Matrix Metalloproteinases and Extracellular Matrix in the Central Nervous System G.A. Rosenberg The University of New Mexico, Albuquerque, NM, United States
INTRODUCTION Brain cells are surrounded by extracellular matrix (ECM) made up of large protein molecules. Interstitial fluid (ISF) moves between the cells and along perivascular spaces, mixing with the cerebrospinal fluid (CSF) to act as the lymph of the brain. Early investigators realized that a mechanism to remove waste products of metabolism and to deliver nutrients to the cells was essential, and they postulated the ISF/CSF, acting as a third circulation, performed this essential function. However, they had no idea of the size of the space and no way to visualize it until early electron micrographs showed a space between the cells, using a method of freeze substitution that preserved the water in the brain, demonstrating an appreciable extracellular space (ECS) . Studies have revealed that the ECM is an essential component of the central nervous system (CNS). Estimated to comprise 15–20% of the brain tissue, its complex role in brain development and injury is beginning to emerge. ECM in the adult CNS is localized to three Primer on Cerebrovascular Diseases, Second Edition http://dx.doi.org/10.1016/B978-0-12-803058-5.00061-8
principal compartments: the basal lamina, the perineuronal nets (PNNs), and the neural interstitial matrix in the parenchyma (http://www.nature.com/nrn/journal/ v14/n10/full/nrn3550.html) (Fig. 61.1) . In the CNS, the basal lamina separates endothelial cells from parenchymal tissue and surrounds the pial surface; it is made up of collagen, laminin–nidogen (also known as entactin) complexes, fibronectin, dystroglycan, and perlecan. The basal lamina is a major site of action of the matrix-degrading metalloproteinases (MMPs), which act on the proteins in the basal lamina to disrupt the blood–brain barrier (BBB). The third component of the ECM is the interstitial matrix consisting of a dense network of proteoglycans, hyaluronan, tenascins, and link proteins. PNNs are a layer of lattice-like matrix that enwraps the surface of the soma and dendrites; they are mainly composed of hyaluronan, chondroitin sulfate proteoglycans (CSPG), link proteins, and tenascin R, and play a direct role in the control of CNS plasticity. Their removal is one way in which plasticity can be reactivated in the adult CNS . © 2017 Elsevier Inc. All rights reserved.