No abstract

Axonal injury is a common cause of neurological dysfunction. Unfortunately, in contrast to axons from the peripheral nervous system, the limited capacity of regeneration of central nervous system (CNS) axons is a major obstacle for functional recovery in patients suffering neurological diseases that involve the subcortical white matter. Urokinase-type plasminogen activator (uPA) is a serine proteinase that upon binding to the urokinase-type plasminogen activator receptor (uPAR) catalyzes the conversion of plasminogen into plasmin on the cell surface. uPAR expression increases after an injury, and signaling through uPAR promotes tissue remodeling. However, it is yet unknown whether uPA binding to uPAR has an effect on axonal recovery in the CNS. Here, we used and models of CNS axonal injury to test the hypothesis that uPA binding to uPAR promotes axonal regeneration in the CNS. We found that newly formed growth cones from axons re-emerging from an axonal injury express uPAR and that binding of uPA to this uPAR promotes axonal recovery by a mechanism that does not require the generation of plasmin. Our data indicate that the binding of recombinant uPA or endogenous uPA to uPAR induces membrane recruitment and activation of β1 integrin via the low density lipoprotein receptor-related protein-1 (LRP1), which leads to activation of the Rho family small GTPase Rac1 and Rac1-induced axonal regeneration. Our results show that the uPA/uPAR/LRP1 system is a potential target for the development of therapeutic strategies to promote axonal recovery following a CNS injury.

Heterozygous, loss-of-function mutations in the granulin gene (GRN) encoding progranulin (PGRN) are a common cause of frontotemporal dementia (FTD). Homozygous GRN mutations cause neuronal ceroid lipofuscinosis-11 (CLN11), a lysosome storage disease. PGRN is a secreted glycoprotein that can be proteolytically cleaved into seven bioactive 6 kDa granulins. However, it is unclear how deficiency of PGRN and granulins causes neurodegeneration. To gain insight into the mechanisms of FTD pathogenesis, we utilized Tandem Mass Tag isobaric labeling mass spectrometry to perform an unbiased quantitative proteomic analysis of whole-brain tissue from wild type (Grn+/+) and Grn knockout (Grn−/−) mice at 3- and 19-months of age. At 3-months lysosomal proteins (i.e. Gns, Scarb2, Hexb) are selectively increased indicating lysosomal dysfunction is an early consequence of PGRN deficiency. Additionally, proteins involved in lipid metabolism (Acly, Apoc3, Asah1, Gpld1, Ppt1, and Naaa) are decreased; suggesting lysosomal degradation of lipids may be impaired in the Grn−/− brain. Systems biology using weighted correlation network analysis (WGCNA) of the Grn−/− brain proteome identified 26 modules of highly co-expressed proteins. Three modules strongly correlated to Grn deficiency and were enriched with lysosomal proteins (Gpnmb, CtsD, CtsZ, and Tpp1) and inflammatory proteins (Lgals3, GFAP, CD44, S100a, and C1qa). We find that lysosomal dysregulation is exacerbated with age in the Grn−/− mouse brain leading to neuroinflammation, synaptic loss, and decreased markers of oligodendrocytes, myelin, and neurons. In particular, GPNMB and LGALS3 (galectin-3) were upregulated by microglia and elevated in FTD-GRN brain samples, indicating common pathogenic pathways are dysregulated in human FTD cases and Grn−/− mice. GPNMB levels were significantly increased in the cerebrospinal fluid of FTD-GRN patients, but not in MAPT or C9orf72 carriers, suggesting GPNMB could be a biomarker specific to FTD-GRN to monitor disease onset, progression, and drug response. Our findings support the idea that insufficiency of PGRN and granulins in humans causes neurodegeneration through lysosomal dysfunction, defects in autophagy, and neuroinflammation, which could be targeted to develop effective therapies.

Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin on the cell surface. Our previous studies indicate that uPA and uPAR expression increase in the ischemic brain during the recovery phase from an acute ischemic injury and that uPA binding to uPAR promotes neurological recovery after an acute ischemic stroke. Here, we used male mice genetically deficient on either uPA (uPA) or uPAR (uPAR) or with a four-amino acid substitution into the growth factor domain of uPA that abrogates its binding to uPAR () to investigate the mechanism whereby uPA promotes neurorepair in the ischemic brain. We found that neurons release uPA and astrocytes recruit uPAR to their plasma membrane during the recovery phase from a hypoxic injury and that binding of neuronal uPA to astrocytic uPAR induces astrocytic activation by a mechanism that does not require plasmin generation, but instead is mediated by extracellular signal-regulated kinase 1/2 (ERK1/2)-regulated phosphorylation of the signal transducer and activator of transcription 3 (STAT3). We report that uPA/uPAR binding is necessary and sufficient to induce astrocytic activation in the ischemic brain and that astrocytes activated by neuronal uPA promote synaptic recovery in neurons that have suffered an acute hypoxic injury via a mechanism mediated by astrocytic thrombospondin-1 (TSP1) and synaptic low-density lipoprotein receptor-related protein-1 (LRP1). In summary, we show that uPA/uPAR-induced astrocytic activation mediates a cross talk between astrocytes and injured neurons that promotes synaptic recovery in the ischemic brain. To date, there is no therapeutic strategy to promote synaptic recovery in the injured brain. Here, we show that neurons release urokinase-type plasminogen activator (uPA) and astrocytes recruit the uPA receptor (uPAR) to their plasma membrane during the recovery phase from a hypoxic injury. We found that binding of neuronal uPA to astrocytic uPAR promotes astrocytic activation and that astrocytes activated by uPA-uPAR binding promote synaptic recovery in neurons that have suffered a hypoxic injury by a mechanism that does not require plasmin generation, but instead is mediated by ERK1/2-regulated STAT3 phosphorylation, astrocytic thrombospondin-1 (TSP1) and synaptic low-density lipoprotein receptor-related protein-1 (LRP1). Our work unveils a new biological function for uPA-uPAR as mediator of a neuron-astrocyte cross talk that promotes synaptic recovery in the ischemic brain.

The Runx2 factor is an essential component of the regulatory mechanisms that control transcription during skeletogenesis. Runx2/p57 expression in osteoblastic cells is controlled by the P1 promoter, which is recognized by key regulators of osteoblast differentiation including homeodomain factors and Wnt- and BMP-signaling mediators. Here, we report that the transcription factor C/EBPβ up-regulates Runx2/p57 expression by directly binding to the Runx2 P1 promoter in mesenchymal, pre-osteoblastic and osteoblastic cells. This C/EBPβ-mediated up-regulation is principally dependent on C/EBP site II that is located within the first 180 bp of the proximal P1 promoter region and is highly conserved among mouse, rat, and human Runx2 genes. Our studies reveal how the C/EBPβ factor, known to have a key role during osteogenesis, contributes to regulating the expression of Runx2, the master regulator of osteoblast differentiation.