Tissue Plasminogen Activator Signaling in the Normal and Diseased Brain

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 (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 calcium-mediated 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 α ₂ -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.

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.

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