Tissue Plasminogen Activator in Central Nervous System Physiology and Pathology: From Synaptic Plasticity to Alzheimer's Disease.

Tissue plasminogen activator's (tPA) fibrinolytic function in the vasculature is well-established. This specific role for tPA in the vasculature, however, contrasts with its pleiotropic activities in the central nervous system. Numerous physiological and pathological functions have been attributed to tPA in the central nervous system, including neurite outgrowth and regeneration; synaptic and spine plasticity; neurovascular coupling; neurodegeneration; microglial activation; and blood-brain barrier permeability. In addition, multiple substrates, both plasminogen-dependent and -independent, have been proposed to be responsible for tPA's action(s) in the central nervous system. This review aims to dissect a subset of these different functions and the different molecular mechanisms attributed to tPA in the context of learning and memory. We start from the original research that identified tPA as an immediate-early gene with a putative role in synaptic plasticity to what is currently known about tPA's role in a learning and memory disorder, Alzheimer's disease. We specifically focus on studies demonstrating tPA's involvement in the clearance of amyloid-ß and neurovascular coupling. In addition, given that tPA has been shown to regulate blood-brain barrier permeability, which is perturbed in Alzheimer's disease, this review also discusses tPA-mediated vascular dysfunction and possible alternative mechanisms of action for tPA in Alzheimer's disease pathology.

Characterization of Tissue Plasminogen Activator Expression and Trafficking in the Adult Murine Brain.

Tissue plasminogen activator (tPA) is an immediate-early gene important for regulating physiological processes like synaptic plasticity and neurovascular coupling. It has also been implicated in several pathological processes including blood-brain barrier (BBB) permeability, seizure progression, and stroke. These varied reports suggest that tPA is a pleiotropic mediator whose actions are highly compartmentalized in space and time. The specific localization of tPA, therefore, can provide useful information about its function. Accordingly, the goal of this study was to provide a detailed characterization of tPA's regional, cellular, and subcellular localization in the brain. To achieve this, two new transgenic mouse lines were utilized: (1) a PlatßGAL reporter mouse, which houses the ß-galactosidase gene in the tPA locus and (2) a tPABAC-Cerulean mouse, which has a cerulean-fluorescent protein fused in-frame to the tPA C-terminus. Using these two transgenic reporters, we show that while tPA is expressed throughout most regions of the adult murine brain, it appears to be preferentially targeted to fiber tracts in the limbic system. In the hippocampus, confocal microscopy revealed tPA-Cerulean (tPA-Cer) puncta localized to giant mossy fiber boutons (MFBs) and astrocytes in stratum lucidum. With amplification of the tPA-Cer signal, somatically localized tPA was also observed in the stratum oriens (SO)/alveus layer of both CA1 and CA3 subfields. Coimmunostaining of tPA-Cer and interneuronal markers indicates that these tPA-positive cell bodies belong to a subclass of somatostatin (SST)/oriens-lacunosum moleculare (O-LM) interneurons. Together, these data imply that tPA's localization is differentially regulated, suggesting that its neuromodulatory effects may be compartmentalized and specialized to cell type.

Secondary phosphorylation in myocytes expressing FLAG-tagged and non-tagged phospho-mimetic cardiac troponin I.

Secondary phosphorylation develops in myocytes expressing phospho-mimetic cardiac troponin I (cTnI) but it is not known whether multiple substitutions (e.g. cTnISDTD and cTnIS4D) cause preferential phosphorylation of the remaining endogenous or the phospho-mimetic cTnI in intact myocytes. Western analysis was performed to determine whether the FLAG/total cTnI ratios are similar for phosphorylated versus total cTnI in myocytes expressing phospho-mimetic cTnI with Asp(D) substitutions at S43/45 plus S23/24 (cTnIS4D) or T144 (cTnISDTD). Representative Western analysis of phosphorylated S23/24 (p-S23/24) and S150 (p-S150) are presented along with re-probes using an antibody which detects all cTnI (MAB1691 Ab). The level of p-S150 also is compared to results obtained using single S43D and/or S45D phospho-mimetic substitutions. These results are discussed in more detail in Lang et al. [1].

Microglial-mediated PDGF-CC activation increases cerebrovascular permeability during ischemic stroke.

Treatment of acute ischemic stroke with the thrombolytic tissue plasminogen activator (tPA) can significantly improve neurological outcomes; however, thrombolytic therapy is associated with an increased risk of intra-cerebral hemorrhage (ICH). Previously, we demonstrated that during stroke tPA acting on the parenchymal side of the neurovascular unit (NVU) can increase blood-brain barrier (BBB) permeability and ICH through activation of latent platelet-derived growth factor-CC (PDGF-CC) and signaling by the PDGF receptor-α (PDGFRα). However, in vitro, activation of PDGF-CC by tPA is very inefficient and the mechanism of PDGF-CC activation in the NVU is not known. Here, we show that the integrin Mac-1, expressed on brain microglia/macrophages (denoted microglia throughout), acts together with the endocytic receptor LRP1 in the NVU to promote tPA-mediated activation of PDGF-CC. Mac-1-deficient mice (Mac-1-/-) are protected from tPA-induced BBB permeability but not from permeability induced by intracerebroventricular injection of active PDGF-CC. Immunofluorescence analysis demonstrates that Mac-1, LRP1, and the PDGFRα all localize to the NVU of arterioles, and following middle cerebral artery occlusion (MCAO) Mac-1-/- mice show significantly less PDGFRα phosphorylation, BBB permeability, and infarct volume compared to wild-type mice. Bone-marrow transplantation studies indicate that resident CD11b+ cells, but not bone-marrow-derived leukocytes, mediate the early activation of PDGF-CC by tPA after MCAO. Finally, using a model of thrombotic stroke with late thrombolysis, we show that wild-type mice have an increased incidence of spontaneous ICH following thrombolysis with tPA 5 h after MCAO, whereas Mac-1-/- mice are resistant to the development of ICH even with late tPA treatment. Together, these results indicate that Mac-1 and LRP1 act as co-factors for the activation of PDGF-CC by tPA in the NVU, and suggest a novel mechanism for tightly regulating PDGFRα signaling in the NVU and controlling BBB permeability.

Functional communication between PKC-targeted cardiac troponin I phosphorylation sites.

Increased protein kinase C (PKC) activity is associated with heart failure, and can target multiple cardiac troponin I (cTnI) residues in myocytes, including S23/24, S43/45 and T144. In earlier studies, cTnI-S43D and/or -S45D augmented S23/24 and T144 phosphorylation, which suggested there is communication between clusters. This communication is now explored by evaluating the impact of phospho-mimetic cTnI S43/45D combined with S23/24D (cTnIS4D) or T144D (cTnISDTD). Gene transfer of epitope-tagged cTnIS4D and cTnISDTD into adult cardiac myocytes progressively replaced endogenous cTnI. Partial replacement with cTnISDTD or cTnIS4D accelerated the time to peak (TTP) shortening and time to 50% re-lengthening (TTR50%) on day 2, but peak shortening was only diminished by cTnIS4D. Extensive cTnIS4D replacement continued to accelerate TTP, and decrease shortening amplitude, while TTR50% returned to baseline levels on day 4. In contrast, cTnISDTD modestly reduced shortening amplitude and continued to accelerate myocyte TTP and TTR50%. These results indicate cTnIS43/45 communicates with S23/24 and T144, with S23/24 exacerbating and T144 attenuating the S43/45D-dependent functional deficit. In addition, more severe functional alterations in cTnIS4D myocytes were accompanied by higher levels of secondary phosphorylation compared to cTnISDTD. These results suggest that secondary phosphorylation helps to maintain steady-state contractile function during chronic cTnI phosphorylation at PKC sites.

Functionally conservative substitutions at cardiac troponin I S43/45.

A phospho-null Ala substitution at protein kinase C (PKC)-targeted cardiac troponin I (cTnI) S43/45 reduces myocyte and cardiac contractile function. The goal of the current study was to test whether cTnIS43/45N is an alternative, functionally conservative substitution in cardiac myocytes. Partial and more extensive endogenous cTnI replacement was similar at 2 and 4 days after gene transfer, respectively, for epitope-tagged cTnI and cTnIS43/45N. This replacement did not significantly change thin filament stoichiometry. In functional studies, there were no significant changes in the amplitude and/or rates of contractile shortening and re-lengthening after this partial (2 days) and extensive (4 days) replacement with cTnIS43/45N. The cTnIS43/45N substitution also was not associated with adaptive changes in the myocyte Ca(2+) transient or in phosphorylation of the protein kinase A and C-targeted cTnIS23/24 site. These results provide evidence that cTnIS43/45N is a functionally conservative substitution, and may be appropriate for use as a phospho-null in rodent models designed for studies on PKC modulation of cardiac performance.

Differential protein expression and basal lamina remodeling in human heart failure.

PURPOSE: A goal of this study was to identify and investigate previously unrecognized components of the remodeling process in the progression to heart failure by comparing protein expression in ischemic failing (F) and nonfailing (NF) human hearts. EXPERIMENTAL DESIGN: Protein expression differences were investigated using multidimensional protein identification and validated by Western analysis. This approach detected basal lamina (BL) remodeling, and further studies analyzed samples for evidence of structural BL remodeling. A rat model of pressure overload (PO) was studied to determine whether nonischemic stressors also produce BL remodeling and impact cellular adhesion. RESULTS: Differential protein expression of collagen IV, laminin α2, and nidogen-1 indicated BL remodeling develops in F versus NF hearts Periodic disruption of cardiac myocyte BL accompanied this process in F, but not NF heart. The rat PO myocardium also developed BL remodeling and compromised myocyte adhesion compared to sham controls. CONCLUSIONS AND CLINICAL RELEVANCE: Differential protein expression and evidence of structural and functional BL alterations develop during heart failure. The compromised adhesion associated with this remodeling indicates a high potential for dysfunctional cellular integrity and tethering in failing myocytes. Therapeutically targeting BL remodeling could slow or prevent the progression of heart disease.

Imatinib treatment reduces brain injury in a murine model of traumatic brain injury.

Current therapies for Traumatic brain injury (TBI) focus on stabilizing individuals and on preventing further damage from the secondary consequences of TBI. A major complication of TBI is cerebral edema, which can be caused by the loss of blood brain barrier (BBB) integrity. Recent studies in several CNS pathologies have shown that activation of latent platelet derived growth factor-CC (PDGF-CC) within the brain can promote BBB permeability through PDGF receptor α (PDGFRα) signaling, and that blocking this pathway improves outcomes. In this study we examine the efficacy for the treatment of TBI of an FDA approved antagonist of the PDGFRα, Imatinib. Using a murine model we show that Imatinib treatment, begun 45 min after TBI and given twice daily for 5 days, significantly reduces BBB dysfunction. This is associated with significantly reduced lesion size 24 h, 7 days, and 21 days after TBI, reduced cerebral edema, determined from apparent diffusion co-efficient (ADC) measurements, and with the preservation of cognitive function. Finally, analysis of cerebrospinal fluid (CSF) from human TBI patients suggests a possible correlation between high PDGF-CC levels and increased injury severity. Thus, our data suggests a novel strategy for the treatment of TBI with an existing FDA approved antagonist of the PDGFRα.

Identification of a neurovascular signaling pathway regulating seizures in mice.

OBJECTIVE: A growing body of evidence suggests that increased blood-brain barrier (BBB) permeability can contribute to the development of seizures. The protease tissue plasminogen activator (tPA) has been shown to promote BBB permeability and susceptibility to seizures. In this study, we examined the pathway regulated by tPA in seizures. METHODS: An experimental model of kainate-induced seizures was used in genetically modified mice, including mice deficient in tPA (tPA (-/-) ), its inhibitor neuroserpin (Nsp (-/-) ), or both (Nsp:tPA (-/-) ), and in mice conditionally deficient in the platelet-derived growth factor receptor alpha (PDGFRα). RESULTS: Compared to wild-type (WT) mice, Nsp (-/-) mice have significantly reduced latency to seizure onset and generalization; whereas tPA (-/-) mice have the opposite phenotype, as do Nsp:tPA (-/-) mice. Furthermore, interventions that maintain BBB integrity delay seizure propagation, whereas osmotic disruption of the BBB in seizure-resistant tPA (-/-) mice dramatically reduces the time to seizure onset and accelerates seizure progression. The phenotypic differences in seizure progression between WT, tPA (-/-) , and Nsp (-/-) mice are also observed in electroencephalogram recordings in vivo, but absent in ex vivo electrophysiological recordings where regulation of the BBB is no longer necessary to maintain the extracellular environment. Finally, we demonstrate that these effects on seizure progression are mediated through signaling by PDGFRα on perivascular astrocytes. INTERPRETATION: Together, these data identify a specific molecular pathway involving tPA-mediated PDGFRα signaling in perivascular astrocytes that regulates seizure progression through control of the BBB. Inhibition of PDGFRα signaling and maintenance of BBB integrity might therefore offer a novel clinical approach for managing seizures.

Independent modulation of contractile performance by cardiac troponin I Ser43 and Ser45 in the dynamic sarcomere.

Protein kinase C (PKC) targets cardiac troponin I (cTnI) S43/45 for phosphorylation in addition to other residues. During heart failure, cTnI S43/45 phosphorylation is elevated, and yet there is ongoing debate about its functional role due, in part, to the emergence of complex phenotypes in animal models. The individual functional influences of phosphorylated S43 and S45 also are not yet known. The present study utilizes viral gene transfer of cTnI with phosphomimetic S43D and/or S45D substitutions to evaluate their individual and combined influences on function in intact adult cardiac myocytes. Partial replacement (≤40%) with either cTnIS43D or cTnIS45D reduced the amplitude of contraction, and cTnIS45D slowed contraction and relaxation rates, while there were no significant changes in function with cTnIS43/45D. More extensive replacement (≥70%) with cTnIS43D, cTnIS45D, and cTnIS43/45D each reduced the amplitude of contraction. Additional experiments also showed cTnIS45D reduced myofilament Ca(2+) sensitivity of tension. At the same time, shortening rates returned toward control values with cTnIS45D and the later stages of relaxation also became accelerated in myocytes expressing cTnIS43D and/or S45D. Further studies demonstrated this behavior coincided with adaptive changes in myofilament protein phosphorylation. Taken together, the results observed in myocytes expressing cTnIS43D and/or S45D suggest these 2 residues reduce function via independent mechanism(s). The changes in function associated with the onset of adaptive myofilament signaling suggest the sarcomere is capable of fine tuning PKC-mediated cTnIS43/45 phosphorylation and contractile performance. This modulatory behavior also provides insight into divergent phenotypes reported in animal models with cTnI S43/45 phosphomimetic substitutions.

Agonist activated PKCßII translocation and modulation of cardiac myocyte contractile function.

Elevated protein kinase C ßII (PKCßII) expression develops during heart failure and yet the role of this isoform in modulating contractile function remains controversial. The present study examines the impact of agonist-induced PKCßII activation on contractile function in adult cardiac myocytes. Diminished contractile function develops in response to low dose phenylephrine (PHE, 100 nM) in controls, while function is preserved in response to PHE in PKCßII-expressing myocytes. PHE also caused PKCßII translocation and a punctate distribution pattern in myocytes expressing this isoform. The preserved contractile function and translocation responses to PHE are blocked by the inhibitor, LY379196 (30 nM) in PKCßII-expressing myocytes. Further analysis showed downstream protein kinase D (PKD) phosphorylation and phosphatase activation are associated with the LY379196-sensitive contractile response. PHE also triggered a complex pattern of end-target phosphorylation in PKCßII-expressing myocytes. These patterns are consistent with bifurcated activation of downstream signaling activity by PKCßII.

PKCßII modulation of myocyte contractile performance.

Significant up-regulation of the protein kinase Cß(II) (PKCß(II)) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCß(II) modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCß(II) protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCß(II) was distributed in a peri-nuclear pattern and this expression resulted in diminished rates and amplitude of shortening and re-lengthening compared to controls and myocytes expressing dominant negative PKCß(II) (PKCßDN). Similar decreases were observed in the Ca(2+) transient and the Ca(2+) decay rate slowed in response to caffeine in PKCß(II)-expressing myocytes. Parallel phosphorylation studies indicated PKCß(II) targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCß inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCß(II) expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCß(II) increased Ca(2+)-activated, calmodulin-dependent kinase IIδ (CaMKIIδ) expression and enhanced both CaMKIIδ and protein kinase D (PKD) phosphorylation. Phosphorylation of both signaling targets also was resistant to acute inhibition by LY. These later results provide evidence PKCß(II) modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes.