Axon-derived PACSIN1 binds to the Schwann cell survival receptor, LRP1, and transactivates TrkC to promote gliatrophic activities.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following PNS injury. The ligands and receptors that activate and sustain SC transformation remain incompletely understood. Proteins released by injured axons represent important candidates for activating the SC Repair Program. The low-density lipoprotein receptor-related protein-1 (LRP1) is acutely up-regulated in SCs in response to injury, activating c-Jun, and promoting SC survival. To identify novel LRP1 ligands released in PNS injury, we applied a discovery-based approach in which extracellular proteins in the injured nerve were captured using Fc-fusion proteins containing the ligand-binding motifs of LRP1 (CCR2 and CCR4). An intracellular neuron-specific protein, Protein Kinase C and Casein Kinase Substrate in Neurons (PACSIN1) was identified and validated as an LRP1 ligand. Recombinant PACSIN1 activated c-Jun and ERK1/2 in cultured SCs. Silencing Lrp1 or inhibiting the LRP1 cell-signaling co-receptor, the NMDA-R, blocked the effects of PACSIN1 on c-Jun and ERK1/2 phosphorylation. Intraneural injection of PACSIN1 into crush-injured sciatic nerves activated c-Jun in wild-type mice, but not in mice in which Lrp1 is conditionally deleted in SCs. Transcriptome profiling of SCs revealed that PACSIN1 mediates gene expression events consistent with transformation to the repair phenotype. PACSIN1 promoted SC migration and viability following the TNFα challenge. When Src family kinases were pharmacologically inhibited or the receptor tyrosine kinase, TrkC, was genetically silenced or pharmacologically inhibited, PACSIN1 failed to induce cell signaling and prevent SC death. Collectively, these studies demonstrate that PACSIN1 is a novel axon-derived LRP1 ligand that activates SC repair signaling by transactivating TrkC.

Establishing priorities for implementation of large language models in pathology and laboratory medicine.

Artificial intelligence and machine learning have numerous applications in pathology and laboratory medicine. The release of ChatGPT prompted speculation regarding the potentially transformative role of large-language models (LLMs) in academic pathology, laboratory medicine, and pathology education. Because of the potential to improve LLMs over the upcoming years, pathology and laboratory medicine clinicians are encouraged to embrace this technology, identify pathways by which LLMs may support our missions in education, clinical practice, and research, participate in the refinement of AI modalities, and design user-friendly interfaces that integrate these tools into our most important workflows. Challenges regarding the use of LLMs, which have already received considerable attention in a general sense, are also reviewed herein within the context of the pathology field and are important to consider as LLM applications are identified and operationalized.

An LRP1-binding motif in cellular prion protein replicates cell-signaling activities of the full-length protein.

Low-density lipoprotein receptor-related protein-1 (LRP1) functions as a receptor for nonpathogenic cellular prion protein (PrPC), which is released from cells by ADAM (a disintegrin and metalloproteinase domain) proteases or in extracellular vesicles. This interaction activates cell signaling and attenuates inflammatory responses. We screened 14-mer PrPC-derived peptides and identified a putative LRP1 recognition motif in the PrPC sequence spanning residues 98-111. A synthetic peptide (P3) corresponding to this region replicated the cell-signaling and biological activities of full-length shed PrPC. P3 blocked LPS-elicited cytokine expression in macrophages and microglia and rescued the heightened sensitivity to LPS in mice in which the PrPC gene (Prnp) had been deleted. P3 activated ERK1/2 and induced neurite outgrowth in PC12 cells. The response to P3 required LRP1 and the NMDA receptor and was blocked by the PrPC-specific antibody, POM2. P3 has Lys residues, which are typically necessary for LRP1 binding. Converting Lys100 and Lys103 into Ala eliminated the activity of P3, suggesting that these residues are essential in the LRP1-binding motif. A P3 derivative in which Lys105 and Lys109 were converted into Ala retained activity. We conclude that the biological activities of shed PrPC, attributed to interaction with LRP1, are retained in synthetic peptides, which may be templates for therapeutics development.

An antibody that targets cell-surface glucose-regulated protein-78 inhibits expression of inflammatory cytokines and plasminogen activator inhibitors by macrophages.

Glucose-regulated protein-78 (Grp78) is an endoplasmic reticulum chaperone, which is secreted by cells and associates with cell surfaces, where it functions as a receptor for activated α2 -macroglobulin (α2 M) and tissue-type plasminogen activator (tPA). In macrophages, α2 M and tPA also bind to the transmembrane receptor, LDL receptor-related protein-1 (LRP1), activating a cell-signaling receptor assembly that includes the NMDA receptor (NMDA-R) to suppress innate immunity. Herein, we demonstrate that an antibody targeting Grp78 (N88) inhibits NFκB activation and expression of proinflammatory cytokines in bone marrow-derived macrophages (BMDMs) treated with the toll-like receptor-4 (TLR4) ligand, lipopolysaccharide, or with agonists that activate TLR2, TLR7, or TLR9. Pharmacologic inhibition of the NMDA-R or deletion of the gene encoding LRP1 (Lrp1) in BMDMs neutralizes the activity of N88. The fibrinolysis protease inhibitor, plasminogen activator inhibitor-1 (PAI1), has been implicated in diverse diseases including metabolic syndrome, cardiovascular disease, and type 2 diabetes. Deletion of Lrp1 independently increased expression of PAI1 and PAI2 in BMDMs, as did treatment of wild-type BMDMs with TLR agonists. tPA, α2 M, and N88 inhibited expression of PAI1 and PAI2 in BMDMs treated with TLR-activating agents. Inhibiting Src family kinases blocked the ability of both N88 and tPA to function as anti-inflammatory agents, suggesting that the cell-signaling pathway activated by tPA and N88, downstream of LRP1 and the NMDA-R, may be equivalent. We conclude that targeting cell-surface Grp78 may be effective in suppressing innate immunity by a mechanism that requires LRP1 and the NMDA-R.

The LRP1/CD91 ligands, tissue-type plasminogen activator, α2-macroglobulin, and soluble cellular prion protein have distinct co-receptor requirements for activation of cell-signaling.

LDL Receptor-related Protein-1 (LRP1/CD91) binds diverse ligands, many of which activate cell-signaling. Herein, we compared three LRP1 ligands that inhibit inflammatory responses triggered by lipopolysaccharide (LPS), including: enzymatically-inactive tissue-type plasminogen activator (EI-tPA); activated α2-macroglobulin (α2M); and S-PrP, a soluble derivative of nonpathogenic cellular prion protein (PrPC). In bone marrow-derived macrophages, the N-methyl-D-aspartate receptor was essential for all three LRP1 ligands to activate cell-signaling and inhibit LPS-induced cytokine expression. Intact lipid rafts also were essential. Only α2M absolutely required LRP1. LRP1 decreased the EI-tPA concentration required to activate cell-signaling and antagonize LPS but was not essential, mimicking its role as a S-PrP co-receptor. Membrane-anchored PrPC also functioned as a co-receptor for EI-tPA and α2M, decreasing the ligand concentration required for cell-signaling and LPS antagonism; however, when the concentration of EI-tPA or α2M was sufficiently increased, cell-signaling and LPS antagonism occurred independently of PrPC. S-PrP is the only LRP1 ligand in this group that activated cell-signaling independently of membrane-anchored PrPC. EI-tPA, α2M, and S-PrP inhibited LPS-induced LRP1 shedding from macrophages, a process that converts LRP1 into a pro-inflammatory product. Differences in the co-receptors required for anti-inflammatory activity may explain why LRP1 ligands vary in ability to target macrophages in different differentiation states.

Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases1-3. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing4,5. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Wastewater sequencing uncovers early, cryptic SARS-CoV-2 variant transmission.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing/sequencing capacity, which can also introduce biases. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here, we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We develop and deploy improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detect emerging variants of concern up to 14 days earlier in wastewater samples, and identify multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Cellular prion protein in human plasma-derived extracellular vesicles promotes neurite outgrowth via the NMDA receptor-LRP1 receptor system.

Exosomes and other extracellular vesicles (EVs) participate in cell-cell communication. Herein, we isolated EVs from human plasma and demonstrated that these EVs activate cell signaling and promote neurite outgrowth in PC-12 cells. Analysis of human plasma EVs purified by sequential ultracentrifugation using tandem mass spectrometry indicated the presence of multiple plasma proteins, including α2-macroglobulin, which is reported to regulate PC-12 cell physiology. We therefore further purified EVs by molecular exclusion or phosphatidylserine affinity chromatography, which reduced plasma protein contamination. EVs subjected to these additional purification methods exhibited unchanged activity in PC-12 cells, even though α2-macroglobulin was reduced to undetectable levels. Nonpathogenic cellular prion protein (PrPC) was carried by human plasma EVs and essential for the effects of EVs on PC-12 cells, as EV-induced cell signaling and neurite outgrowth were blocked by the PrPC-specific antibody, POM2. In addition, inhibitors of the N-methyl-d-aspartate (NMDA) receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1) blocked the effects of plasma EVs on PC-12 cells, as did silencing of Lrp1 or the gene encoding the GluN1 NMDA-R subunit (Grin1). These results implicate the NMDA-R-LRP1 complex as the receptor system responsible for mediating the effects of EV-associated PrPC. Finally, EVs harvested from rat astrocytes carried PrPC and replicated the effects of human plasma EVs on PC-12 cell signaling. We conclude that interaction of EV-associated PrPC with the NMDA-R-LRP1 complex in target cells represents a novel mechanism by which EVs may participate in intercellular communication in the nervous system.

A Soluble PrPC Derivative and Membrane-Anchored PrPC in Extracellular Vesicles Attenuate Innate Immunity by Engaging the NMDA-R/LRP1 Receptor Complex.

Nonpathogenic cellular prion protein (PrPC) demonstrates anti-inflammatory activity; however, the responsible mechanisms are incompletely defined. PrPC exists as a GPI-anchored membrane protein in diverse cells; however, PrPC may be released from cells by ADAM proteases or when packaged into extracellular vesicles (EVs). In this study, we show that a soluble derivative of PrPC (S-PrP) counteracts inflammatory responses triggered by pattern recognition receptors in macrophages, including TLR2, TLR4, TLR7, TLR9, NOD1, and NOD2. S-PrP also significantly attenuates the toxicity of LPS in mice. The response of macrophages to S-PrP is mediated by a receptor assembly that includes the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1). PrPC was identified in EVs isolated from human plasma. These EVs replicated the activity of S-PrP, inhibiting cytokine expression and IκBα phosphorylation in LPS-treated macrophages. The effects of plasma EVs on LPS-treated macrophages were blocked by PrPC-specific Ab, by antagonists of LRP1 and the NMDA-R, by deleting Lrp1 in macrophages, and by inhibiting Src family kinases. Phosphatidylinositol-specific phospholipase C dissociated the LPS-regulatory activity from EVs, rendering the EVs inactive as LPS inhibitors. The LPS-regulatory activity that was lost from phosphatidylinositol-specific phospholipase C-treated EVs was recovered in solution. Collectively, these results demonstrate that GPI-anchored PrPC is the essential EV component required for the observed immune regulatory activity of human plasma EVs. S-PrP and EV-associated PrPC regulate innate immunity by engaging the NMDA-R/LRP1 receptor system in macrophages. The scope of pattern recognition receptors antagonized by S-PrP suggests that released forms of PrPC may have broad anti-inflammatory activity.

Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy.

Schwann cells (SCs) are known to produce extracellular vesicles (EV) that participate in cell-cell communication by transferring cargo to target cells, including mRNAs, microRNAs, and biologically active proteins. Herein, we report a novel mechanism whereby SC EVs may regulate PNS physiology, especially in injury, by controlling the activity of TNFα. SCs actively sequester tumor necrosis factor receptor-1 (TNFR1) into EVs at high density, accounting for about 2% of the total protein in SC EVs (~1000 copies TNFR1/EV). Although TNFR2 was robustly expressed by SCs in culture, TNFR2 was excluded from SC EVs. SC EV TNFR1 bound TNFα, decreasing the concentration of free TNFα available to bind to cells and thus served as a TNFα decoy. SC EV TNFR1 significantly inhibited TNFα-induced p38 MAPK phosphorylation in cultured SCs. When TNFR1 was proteolytically removed from SC EVs using tumor necrosis factor-α converting enzyme (TACE) or neutralized with antibody, the ability of TNFα to activate p38 MAPK in the presence of these EVs was restored. As further evidence of its decoy activity, SC EV TNFR1 modified TNFα activities in vitro including: (1) regulation of expression of other cytokines; (2) effects on SC morphology; and (3) effects on SC viability. SC EVs also modified the effects of TNFα on sciatic nerve morphology and neuropathic pain-related behavior in vivo. By sequestering TNFR1 in EVs, SCs may buffer against the potentially toxic effects of TNFα. SC EVs provide a novel mechanism for the spatial and temporal regulation of neuro-inflammation.

Plasminogen activator receptor assemblies in cell signaling, innate immunity, and inflammation.

Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are serine proteases and major activators of fibrinolysis in mammalian systems. Because fibrinolysis is an essential component of the response to tissue injury, diverse cells, including cells that participate in the response to injury, have evolved receptor systems to detect tPA and uPA and initiate appropriate cell-signaling responses. Formation of functional receptor systems for the plasminogen activators requires assembly of diverse plasma membrane proteins, including but not limited to: the urokinase receptor (uPAR); integrins; N-formyl peptide receptor-2 (FPR2), receptor tyrosine kinases (RTKs), the N-methyl-d-aspartate receptor (NMDA-R), and low-density lipoprotein receptor-related protein-1 (LRP1). The cell-signaling responses elicited by tPA and uPA impact diverse aspects of cell physiology. This review describes rapidly evolving knowledge regarding the structure and function of plasminogen activator receptor assemblies. How these receptor assemblies regulate innate immunity and inflammation is then considered.

SARS-CoV-2 Serology Status Detected by Commercialized Platforms Distinguishes Previous Infection and Vaccination Adaptive Immune Responses.

BACKGROUND: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 110 million individuals and led to 2.5 million deaths worldwide. As more individuals are vaccinated, the clinical performance and utility of SARS-CoV-2 serology platforms needs to be evaluated. METHODS: The ability of 4 commercial SARS-CoV-2 serology platforms to detect previous infection or vaccination were evaluated using a cohort of 53 patients who were SARS-CoV-2 PCR positive, 89 SARS-CoV-2-vaccinated healthcare workers (Pfizer or Moderna), and 127 patients who were SARS-CoV-2 negative. Serology results were compared to a cell-based SARS-CoV-2 pseudovirus (PSV) neutralizing antibodies assay. RESULTS: The Roche S-(spike) antibody and Diazyme neutralizing antibodies (NAbs) assays detected adaptive immune response in 100.0% and 90.1% of vaccinated individuals who received 2 doses of vaccine (initial and booster), respectively. The Roche N-(nucleocapsid) antibody assay and Diazyme IgG assay did not detect adaptive immune response in vaccinated individuals. The Diazyme NAbs assay correlated with the PSV SARS-CoV-2 median infective dose (ID50) neutralization titers (R2 = 0.70), while correlation of the Roche S-antibody assay was weaker (R2 = 0.39). Median PSV SARS-CoV-2 ID50 titers more than doubled in vaccinated individuals who received 2 doses of the Moderna vaccine (ID50, 597) compared to individuals who received a single dose (ID50, 284). CONCLUSIONS: The Roche S-antibody and Diazyme NAbs assays robustly detected adaptive immune responses in SARS-CoV-2 vaccinated individuals and SARS-CoV-2 infected individuals. The Diazyme NAbs assay strongly correlates with the PSV SARS-CoV-2 NAbs in vaccinated individuals. Understanding the reactivity of commercially available serology platforms is important when distinguishing vaccination response versus natural infection.

SARS-CoV-2 Serology Status Detected by Commercialized Platforms Distinguishes Previous Infection and Vaccination Adaptive Immune Responses.

Background: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 110 million individuals and led to 2.5 million deaths worldwide. As more individuals are vaccinated, the clinical performance and utility of SARS-CoV-2 serology platforms needs to be evaluated. Methods: The ability of four commercial SARS-CoV-2 serology platforms to detect previous infection or vaccination were evaluated using a cohort of 53 SARS-CoV-2 PCR-positive patients, 89 SARS-CoV-2-vaccinated healthcare workers (Pfizer or Moderna), and 127 SARS-CoV-2 negative patients. Serology results were compared to a cell based SARS-CoV-2 pseudovirus (PSV) neutralizing antibodies assay. Results: The Roche S-(spike) antibody and Diazyme neutralizing antibodies (NAbs) assays detected adaptive immune response in 100.0% and 90.1% of vaccinated individuals who received two-doses of vaccine (initial and booster), respectively. The Roche N-(nucleocapsid) antibody assay and Diazyme IgG assay did not detect adaptive immune response in vaccinated individuals. The Diazyme Nabs assay correlated with the PSV SARS-CoV-2 ID50 neutralization titers (R2= 0.70), while correlation of the Roche S-antibody assay was weaker (R2= 0.39). Median PSV SARS-CoV-2 ID50 titers more than doubled in vaccinated individuals who received two-doses of the Moderna vaccine (ID50: 597) compared to individuals that received a single dose (ID50: 284). Conclusions: The Roche S-antibody and Diazyme NAbs assays robustly detected adaptive immune responses in SARS-CoV-2 vaccinated individuals and SARS-CoV-2 infected individuals. The Diazyme NAbs assay strongly correlates with the PSV SARS-CoV-2 NAbs in vaccinated individuals. Understanding the reactivity of commercially available serology platforms is important when distinguishing vaccination response versus natural infection.

Enzymatically Inactive Tissue-Type Plasminogen Activator Reverses Disease Progression in the Dextran Sulfate Sodium Mouse Model of Inflammatory Bowel Disease.

Enzymatically inactive tissue-type plasminogen activator (EI-tPA) does not activate fibrinolysis, but interacts with the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1) in macrophages to block innate immune system responses mediated by toll-like receptors. Herein, we examined the ability of EI-tPA to treat colitis in mice, induced by dextran sulfate sodium. In two separate studies, designed to generate colitis of differing severity, a single dose of EI-tPA administered after inflammation established significantly improved disease parameters. EI-tPA-treated mice demonstrated improved weight gain. Stools improved in character and became hemoccult negative. Abdominal tenderness decreased. Colon shortening significantly decreased in EI-tPA-treated mice, suggesting attenuation of irreversible tissue damage and remodeling. Furthermore, histopathologic evidence of disease decreased in the distal 25% of the colon in EI-tPA-treated mice. EI-tPA did not decrease the number of CD45-positive leukocytes or F4/80-positive macrophage-like cells detected in extracts of colons from dextran sulfate sodium-treated mice as assessed by flow cytometry. However, multiple colon cell types expressed the NMDA-R, suggesting the ability of diverse cells, including CD3-positive cells, CD103-positive cells, Ly6G-positive cells, and epithelial cell adhesion molecule-positive epithelial cells to respond to EI-tPA. Mesenchymal cells that line intestinal crypts and provide barrier function expressed LRP1, thereby representing another potential target for EI-tPA. These results demonstrate that the NMDA-R/LRP1 receptor system may be a target for drug development in diseases characterized by tissue damage and chronic inflammation.

Deletion of the Gene Encoding the NMDA Receptor GluN1 Subunit in Schwann Cells Causes Ultrastructural Changes in Remak Bundles and Hypersensitivity in Pain Processing.

Abnormalities in interactions between sensory neurons and Schwann cells (SCs) may result in heightened pain processing and chronic pain states. We previously reported that SCs express the NMDA receptor (NMDA-R), which activates cell signaling in response to glutamate and specific protein ligands, such as tissue-type plasminogen activator. Herein, we genetically targeted grin1 encoding the essential GluN1 NMDA-R subunit, conditionally in SCs, to create a novel mouse model in which SCs are NMDA-R-deficient (GluN1- mice). These mice demonstrated increased sensitivity to light touch, pinprick, and thermal hyperalgesia in the absence of injury, without associated changes in motor function. Ultrastructural analysis of adult sciatic nerve in GluN1- mice revealed increases in the density of Aδ fibers and Remak bundles and a decrease in the density of Aß fibers, without altered g-ratios. Abnormalities in adult Remak bundle ultrastructure were also present including aberrant C-fiber ensheathment, distances between axons, and increased poly-axonal pockets. Developmental and post radial sorting defects contributed to altered nerve fiber densities in adult. Uninjured sciatic nerves in GluN1- mice did not demonstrate an increase in neuroinflammatory infiltrates. Transcriptome profiling of dorsal root ganglia (DRGs) revealed 138 differentially regulated genes in GluN1- mice. One third of the regulated genes are known to be involved in pain processing, including sprr1a, npy, fgf3, atf3, and cckbr, which were significantly increased. The intraepidermal nerve fiber density (IENFD) was significantly decreased in the skin of GluN1- mice. Collectively, these findings demonstrate that SC NMDA-R is essential for normal PNS development and for preventing development of pain states.SIGNIFICANCE STATEMENT Chronic unremitting pain is a prevalent medical condition; however, the molecular mechanisms that underlie heightened pain processing remain incompletely understood. Emerging data suggest that abnormalities in Schwann cells (SCs) may cause neuropathic pain. We established a novel mouse model for small fiber neuropathy (SFN) in which grin1, the gene that encodes the NMDA receptor (NMDA-R) GluN1 subunit, is deleted in SCs. These mice demonstrate hypersensitivity in pain processing in the absence of nerve injury. Changes in the density of intraepidermal small fibers, the ultrastructure of Remak bundles, and the transcriptome of dorsal root ganglia (DRGs) provide possible explanations for the increase in pain processing. Our results support the hypothesis that abnormalities in communication between sensory nerve fibers and SCs may result in pain states.

A soluble derivative of PrPC activates cell-signaling and regulates cell physiology through LRP1 and the NMDA receptor.

Cellular prion protein (PrPC) is a widely expressed glycosylphosphatidylinositol-anchored membrane protein. Scrapie prion protein is a misfolded and aggregated form of PrPC responsible for prion-induced neurodegenerative diseases. Understanding the function of the nonpathogenic PrPC monomer is an important objective. PrPC may be shed from the cell surface to generate soluble derivatives. Herein, we studied a recombinant derivative of PrPC (soluble cellular prion protein, S-PrP) that corresponds closely in sequence to a soluble form of PrPC shed from the cell surface by proteases in the A Disintegrin And Metalloprotease (ADAM) family. S-PrP activated cell-signaling in PC12 and N2a cells. TrkA was transactivated by Src family kinases and extracellular signal-regulated kinase 1/2 was activated downstream of Trk receptors. These cell-signaling events were dependent on the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1), which functioned as a cell-signaling receptor system in lipid rafts. Membrane-anchored PrPC and neural cell adhesion molecule were not required for S-PrP-initiated cell-signaling. S-PrP promoted PC12 cell neurite outgrowth. This response required the NMDA-R, LRP1, Src family kinases, and Trk receptors. In Schwann cells, S-PrP interacted with the LRP1/NMDA-R system to activate extracellular signal-regulated kinase 1/2 and promote cell migration. The effects of S-PrP on PC12 cell neurite outgrowth and Schwann cell migration were similar to those caused by other proteins that engage the LRP1/NMDA-R system, including activated α2-macroglobulin and tissue-type plasminogen activator. Collectively, these results demonstrate that shed forms of PrPC may exhibit important biological activities in the central nervous system and the peripheral nervous system by serving as ligands for the LRP1/NMDA-R system.

Plasminogen Receptors in Human Malignancies: Effects on Prognosis and Feasibility as Targets for Drug Development.

The major proteases that constitute the fibrinolysis system are tightly regulated. Protease inhibitors target plasmin, the protease responsible for fibrin degradation, and the proteases that convert plasminogen into plasmin, including tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). A second mechanism by which fibrinolysis is regulated involves exosite interactions, which localize plasminogen and its activators to fibrin, extracellular matrix (ECM) proteins, and cell surfaces. Once plasmin is generated in association with cell surfaces, it may cleave transmembrane proteins, activate growth factors, release growth factors from ECM proteins, remodel ECM, activate metalloproteases, and trigger cell-signaling by cleaving receptors in the Proteaseactivated Receptor (PAR) family. These processes are all implicated in cancer. It is thus not surprising that a family of structurally diverse but functionally similar cell-surface proteins, called Plasminogen Receptors (PlgRs), which increase the catalytic efficiency of plasminogen activation, have received attention for their possible function in cancer and as targets for anticancer drug development. In this review, we consider four previously described PlgRs, including: α-enolase, annexin-A2, Plg-RKT, and cytokeratin-8, in human cancer. To compare the PlgRs, we mined transcriptome profiling data from The Cancer Genome Atlas (TCGA) and searched for correlations between PlgR expression and patient survival. In glioma, the expression of specific PlgRs correlates with tumor grade. In a number of malignancies, including glioblastoma and liver cancer, increased expression of α-enolase or annexin-A2 is associated with an unfavorable prognosis. Whether these correlations reflect the function of PlgRs as receptors for plasminogen or other activities is discussed.

Fibrinolysis protease receptors promote activation of astrocytes to express pro-inflammatory cytokines.

BACKGROUND: Astrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; however, compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants. Recent studies suggest that the fibrinolysis system may regulate inflammation. The goal of this study was to test whether fibrinolysis system components activate astrocytes and if so, elucidate the responsible biochemical pathway. METHODS: Primary cultures of astrocytes and microglia were prepared from neonatal mouse brains. The ability of purified fibrinolysis system proteins to elicit a pro-inflammatory response was determined by measuring expression of the mRNAs encoding tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and chemokine (C-C motif) ligand 2 (CCL2). IκBα phosphorylation also was measured. Plasminogen activation in association with cells was detected by chromogenic substrate hydrolysis. The activity of specific receptors was tested using neutralizing antibodies and reagents. RESULTS: Astrocytes expressed pro-inflammatory cytokines when treated with plasminogen but not when treated with agonists for Toll-like Receptor-4 (TLR4), TLR2, or TLR9. Microglia also expressed pro-inflammatory cytokines in response to plasminogen; however, in these cells, the response was observed only when tissue-type plasminogen activator (tPA) was added to activate plasminogen. In astrocytes, endogenously produced urokinase-type plasminogen activator (uPA) converted plasminogen into plasmin in the absence of tPA. Plasminogen activation was dependent on the plasminogen receptor, α-enolase, and the uPA receptor, uPAR. Although uPAR is capable of directly activating cell-signaling, the receptor responsible for cytokine expression and IκBα phosphorylation response to plasmin was Protease-activated Receptor-1 (PAR-1). The pathway, by which plasminogen induced astrocyte activation, was blocked by inhibiting any one of the three receptors implicated in this pathway with reagents such as εACA, α-enolase-specific antibody, uPAR-specific antibody, the uPA amino terminal fragment, or a pharmacologic PAR-1 inhibitor. CONCLUSIONS: Plasminogen may activate astrocytes for pro-inflammatory cytokine expression through the concerted action of at least three distinct fibrinolysis protease receptors. The pathway is dependent on uPA to activate plasminogen, which is expressed endogenously by astrocytes in culture but also may be provided by other cells in the astrocytic cell microenvironment in the CNS.

Tissue-type plasminogen activator-primed human iPSC-derived neural progenitor cells promote motor recovery after severe spinal cord injury.

The goal of stem cell therapy for spinal cord injury (SCI) is to restore motor function without exacerbating pain. Induced pluripotent stem cells (iPSC) may be administered by autologous transplantation, avoiding immunologic challenges. Identifying strategies to optimize iPSC-derived neural progenitor cells (hiNPC) for cell transplantation is an important objective. Herein, we report a method that takes advantage of the growth factor-like and anti-inflammatory activities of the fibrinolysis protease, tissue plasminogen activator tPA, without effects on hemostasis. We demonstrate that conditioning hiNPC with enzymatically-inactive tissue-type plasminogen activator (EI-tPA), prior to grafting into a T3 lesion site in a clinically relevant severe SCI model, significantly improves motor outcomes. EI-tPA-primed hiNPC grafted into lesion sites survived, differentiated, acquired markers of motor neuron maturation, and extended ßIII-tubulin-positive axons several spinal segments below the lesion. Importantly, only SCI rats that received EI-tPA primed hiNPC demonstrated significantly improved motor function, without exacerbating pain. When hiNPC were treated with EI-tPA in culture, NMDA-R-dependent cell signaling was initiated, expression of genes associated with stemness (Nestin, Sox2) was regulated, and thrombin-induced cell death was prevented. EI-tPA emerges as a novel agent capable of improving the efficacy of stem cell therapy in SCI.