The Effects of a Physical Activity Intervention on Adiposity, Physical Fitness and Motor Competence: A School-Based, Non-Randomized Controlled Trial.

Evidence suggests that early physical activity interventions are a means of preventing childhood obesity and are more effective when delivered in a school setting and based on the ecological model. Therefore, the present study aims to determine the effect of a multicomponent intervention based on the ecological model on adiposity, physical fitness and motor competence in children aged 4 to 5 years. METHODS: This study is a non-randomized controlled trial involving 173 children from Chile. The intervention was based on an ecological model and consisted of a physical activity program with three simultaneous parts, affecting intra- and interpersonal dimensions. The adiposity index, body mass index and waist circumference were measured. For physical fitness, muscle strength in the lower part, speed/agility and cardiorespiratory fitness were measured. Motor competence was assessed using catching, aiming and dynamic and static balance tests. RESULTS: After the intervention, there was no reduction in adiposity indices; in the intervention group, body mass index increased significantly with a high effect size. The intervention group showed significant differences in physical fitness in the components of muscle strength in the lower part (p = 0.000) and speed/agility (p = 0.002). For motor competence, the intervention group showed significant improvements in most components. CONCLUSIONS: The multicomponent intervention did not reduce adiposity indices; however, it caused significant improvements in the physical fitness and motor competence components, so it seems prudent to continue implementing it, given the benefits that adequate levels of motor competence and physical fitness bring to children's health, both in the short and long term.

Intensity of a Physical Exercise Programme Executed through Immersive Virtual Reality.

Evidence suggests that moderate to vigorous physical activity (MVPA) is necessary for health benefits. Immersive virtual reality is a technology that uses images, sounds, and tactile sensations from a simulated world to encourage healthy behaviours and physical activity. The aims of this research are (1) to determine the duration and intensity of physical activity performed through immersive virtual reality; (2) to determine differences in physical activity intensity according to gender. METHODS: A nonprobabilistic convenience sample composed of 39 university students of physical education pedagogy, who performed, through immersive virtual reality, a physical activity programme composed of three levels that progressively increased in complexity. Physical activity intensity was measured using accelerometers. RESULTS: Of the three levels, the most complex was not the one that produced the most minutes of MVPA. The three levels added up to 08:53 min of MVPA. No significant differences were found when comparing them by sex. CONCLUSIONS: The results of this study suggest that an exercise programme delivered through immersive virtual reality generates MVPA levels, with no major differences between sexes. Further research is needed to confirm the contribution of immersive virtual reality to physical activity.

The Association between Sedentary Behavior, Physical Activity, and Physical Fitness with Body Mass Index and Sleep Time in Chilean Girls and Boys: A Cross-Sectional Study.

BACKGROUND: In recent decades, the school population has undergone behavioral changes that have affected their health and adult life. The current educational scenario presents high levels of sedentary behavior, physical inactivity, low physical fitness, high levels of obesity, and non-compliance with sleep recommendations. In Chile, the scientific evidence on associations between these behaviors is incipient. OBJECTIVE: To analyze the association between sedentary behavior, physical activity, and physical fitness with BMI and minutes of sleep in Chilean children aged 10 to 11 years. METHODS: A non-probabilistic convenience sample of 222 schoolchildren aged 10 to 11 years. The variables measured were body composition (BMI), cardiorespiratory fitness (20 m shuttle run test), lower and upper muscular strength (long jump test and handgrip dynamometry), speed (4 × 10 m running), and flexibility (sit and reach test). Physical activity and sleep were measured by accelerometers. RESULTS: Of the participants, 60.4% and 90.6% did not comply with sleep and physical activity recommendations, respectively. Physical fitness was higher in boys in all components. The results of the linear regression show that in girls, moderate-vigorous-intensity physical activity, lower- and upper-body muscular strength, and cardiorespiratory endurance were associated with BMI and sleep. In boys, light-intensity physical activity and upper-body muscular strength were associated with both variables. CONCLUSIONS: Physical activity intensity, strength, and cardiorespiratory fitness were associated with BMI and sleep; however, physical activity intensity and associated physical fitness components differed by gender.

Granulins rescue inflammation, lysosome dysfunction, and neuropathology in a mouse model of progranulin deficiency.

Progranulin (PGRN) deficiency is linked to neurodegenerative diseases including frontotemporal dementia, Alzheimer's disease, Parkinson's disease, and neuronal ceroid lipofuscinosis. Proper PGRN levels are critical to maintain brain health and neuronal survival, however the function of PGRN is not well understood. PGRN is composed of 7.5 tandem repeat domains, called granulins, and is proteolytically processed into individual granulins inside the lysosome. The neuroprotective effects of full-length PGRN are well-documented, but the role of granulins is still unclear. Here we report, for the first time, that expression of single granulins is sufficient to rescue the full spectrum of disease pathology in mice with complete PGRN deficiency (Grn-/-). Specifically, rAAV delivery of either human granulin-2 or granulin-4 to Grn-/- mouse brain ameliorates lysosome dysfunction, lipid dysregulation, microgliosis, and lipofuscinosis similar to full-length PGRN. These findings support the idea that individual granulins are the functional units of PGRN, likely mediate neuroprotection within the lysosome, and highlight their importance for developing therapeutics to treat FTD-GRN and other neurodegenerative diseases.

Effectiveness of a physical activity intervention on the overweight and obesity of Chilean schoolchildren.

INTRODUCTION: Physical fitness is considered a measure that integrates most of the functions of the human organism that are involved in physical activity. Motor competence is important due to its observed implications in the physical, mental and social development of children and adolescents. These assessments require the existence of updated reference values that allow the categorization of individuals and groups according to levels of physical fitness and motor competence. This paper describes the study protocol to Effectiveness of a Physical Activity Intervention on the Overweight and Obesity of Chilean Schoolchildren, and in this way determine, through the calculation of a representative sample, reference values for physical fitness and motor competence in children aged 4 to 6 years in the region of La Araucanía, Chile. METHODS: children in pre-basic education in transition grades I and II (4 and 5 years old), and those in the first year of basic education (6 years old) will be invited to participate. The sample will be probabilistic. The measures of a randomized controlled trial (registered in ClinicalTrial.gov NCT04194580) will be used. CONCLUSION: the lack of reference values for physical condition and motor competence for children between 6 and 4 years of age in the Araucanía region highlights the need to establish values, which will contribute to improving the health of children of the age group to be worked on.

Urokinase-Type Plasminogen Activator Triggers Wingless/Int1-Independent Phosphorylation of the Low-Density Lipoprotein Receptor-Related Protein-6 in Cerebral Cortical Neurons.

BACKGROUND: Urokinase-type plasminogen activator (uPA) is a serine proteinase found in excitatory synapses located in the II/III and V cortical layers. The synaptic release of uPA promotes the formation of synaptic contacts and the repair of synapses damaged by various forms of injury, and its abundance is decreased in the synapse of Alzheimer's disease (AD) patients. Inactivation of the Wingless/Int1 (Wnt)-ß-catenin pathway plays a central role in the pathogenesis of AD. Soluble amyloid-ß (Aß) prevents the phosphorylation of the low-density lipoprotein receptor-related protein-6 (LRP6), and the resultant inactivation of the Wnt-ß-catenin pathway prompts the amyloidogenic processing of the amyloid-ß protein precursor (AßPP) and causes synaptic loss. OBJECTIVE: To study the role of neuronal uPA in the pathogenesis of AD. METHODS: We used in vitro cultures of murine cerebral cortical neurons, a murine neuroblastoma cell line transfected with the APP-695 Swedish mutation (N2asw), and mice deficient on either plasminogen, or uPA, or its receptor (uPAR). RESULTS: We show that uPA activates the Wnt-ß-catenin pathway in cerebral cortical neurons by triggering the phosphorylation of LRP6 via a plasmin-independent mechanism that does not require binding of Wnt ligands (Wnts). Our data indicate that uPA-induced activation of the Wnt-ß-catenin pathway protects the synapse from the harmful effects of soluble Aß and prevents the amyloidogenic processing of AßPP by inhibiting the expression of ß-secretase 1 (BACE1) and the ensuing generation of Aß40 and Aß42 peptides. CONCLUSION: uPA protects the synapse and antagonizes the inhibitory effect of soluble Aß on the Wnt-ß-catenin pathway by providing an alternative pathway for LRP6 phosphorylation and ß-catenin stabilization.

Proximity-based labeling reveals DNA damage-induced phosphorylation of fused in sarcoma (FUS) causes distinct changes in the FUS protein interactome.

Accumulation of cytoplasmic inclusions containing fused in sarcoma (FUS), an RNA/DNA-binding protein, is a common hallmark of frontotemporal lobar degeneration and amyotrophic lateral sclerosis neuropathology. We have previously shown that DNA damage can trigger the cytoplasmic accumulation of N-terminally phosphorylated FUS. However, the functional consequences of N-terminal FUS phosphorylation are unknown. To gain insight into this question, we utilized proximity-dependent biotin labeling via ascorbate peroxidase 2 aired with mass spectrometry to investigate whether N-terminal phosphorylation alters the FUS protein-protein interaction network (interactome), and subsequently, FUS function. We report the first analysis comparing the interactomes of three FUS variants: homeostatic wildtype FUS (FUS WT), phosphomimetic FUS (FUS PM; a proxy for N-terminally phosphorylated FUS), and the toxic FUS proline 525 to leucine mutant (FUS P525L) that causes juvenile amyotrophic lateral sclerosis. We found that the phosphomimetic FUS interactome is uniquely enriched for a group of cytoplasmic proteins that mediate mRNA metabolism and translation, as well as nuclear proteins involved in the spliceosome and DNA repair functions. Furthermore, we identified and validated the RNA-induced silencing complex RNA helicase MOV10 as a novel interacting partner of FUS. Finally, we provide functional evidence that N-terminally phosphorylated FUS may disrupt homeostatic translation and steady-state levels of specific mRNA transcripts. Taken together, these results highlight phosphorylation as a unique modulator of the interactome and function of FUS.

Tissue-type plasminogen activator induces TNF-α-mediated preconditioning of the blood-brain barrier.

Ischemic tolerance is a phenomenon whereby transient exposure to a non-injurious preconditioning stimulus triggers resistance to a subsequent lethal ischemic insult. Despite the fact that not only neurons but also astrocytes and endothelial cells have a unique response to preconditioning stimuli, current research has been focused mostly on the effect of preconditioning on neuronal death. Thus, it is unclear if the blood-brain barrier (BBB) can be preconditioned independently of an effect on neuronal survival. The release of tissue-type plasminogen activator (tPA) from perivascular astrocytes in response to an ischemic insult increases the permeability of the BBB. In line with these observations, treatment with recombinant tPA increases the permeability of the BBB and genetic deficiency of tPA attenuates the development of post-ischemic edema. Here we show that tPA induces ischemic tolerance in the BBB independently of an effect on neuronal survival. We found that tPA renders the BBB resistant to an ischemic injury by inducing TNF-α-mediated astrocytic activation and increasing the abundance of aquaporin-4-immunoreactive astrocytic end-feet processes in the neurovascular unit. This is a new role for tPA, that does not require plasmin generation, and with potential therapeutic implications for patients with cerebrovascular disease.

Urokinase-type plasminogen activator-mediated crosstalk between N-cadherin and ß-catenin promotes wound healing.

Urokinase-type plasminogen activator (uPA; encoded by Plau) is a serine proteinase that, in the central nervous system, induces astrocytic activation. ß-Catenin is a protein that links the cytoplasmic tail of cadherins to the actin cytoskeleton, thus securing the formation of cadherin-mediated cell adhesion complexes. Disruption of cell-cell contacts leads to the detachment of ß-catenin from cadherins, and ß-catenin is then degraded by the proteasome following its phosphorylation by GSK3ß. Here, we show that astrocytes release uPA following a scratch injury, and that this uPA promotes wound healing via a plasminogen-independent mechanism. We found that uPA induces the detachment of ß-catenin from the cytoplasmic tail of N-cadherin (NCAD; also known as CDH2) by triggering its phosphorylation at Tyr654. Surprisingly, this is not followed by degradation of ß-catenin because uPA also induces the phosphorylation of the low density lipoprotein receptor-related protein 6 (LRP6) at Ser1490, which then blocks the kinase activity of GSK3ß. Our work indicates that the ensuing cytoplasmic accumulation of ß-catenin is followed by its nuclear translocation and ß-catenin-triggered transcription of the receptor for uPA (Plaur), which in turn is required for uPA to induce astrocytic wound healing.

Lysosome dysfunction as a cause of neurodegenerative diseases: Lessons from frontotemporal dementia and amyotrophic lateral sclerosis.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative disorders that are thought to exist on a clinical and pathological spectrum. FTD and ALS are linked by shared genetic causes (e.g. C9orf72 hexanucleotide repeat expansions) and neuropathology, such as inclusions of ubiquitinated, misfolded proteins (e.g. TAR DNA-binding protein 43; TDP-43) in the CNS. Furthermore, some genes that cause FTD or ALS when mutated encode proteins that localize to the lysosome or modulate endosome-lysosome function, including lysosomal fusion, cargo trafficking, lysosomal acidification, autophagy, or TFEB activity. In this review, we summarize evidence that lysosomal dysfunction, caused by genetic mutations (e.g. C9orf72, GRN, MAPT, TMEM106B) or toxic-gain of function (e.g. aggregation of TDP-43 or tau), is an important pathogenic disease mechanism in FTD and ALS. Further studies into the normal function of many of these proteins are required and will help uncover the mechanisms that cause lysosomal dysfunction in FTD and ALS. Mutations or polymorphisms in genes that encode proteins important for endosome-lysosome function also occur in other age-dependent neurodegenerative diseases, including Alzheimer's (e.g. APOE, PSEN1, APP) and Parkinson's (e.g. GBA, LRRK2, ATP13A2) disease. A more complete understanding of the common and unique features of lysosome dysfunction across the spectrum of neurodegeneration will help guide the development of therapies for these devastating diseases.

Urokinase-type plasminogen activator promotes N-cadherin-mediated synaptic recovery in the ischemic brain.

Urokinase-type plasminogen activator (uPA) is a serine proteinase that catalyzes the generation of plasmin on the cell surface and activates cell signaling pathways that promote remodeling and repair. Neuronal cadherin (NCAD) is a transmembrane protein that in the mature brain mediates the formation of synaptic contacts in the II/III and V cortical layers. Our studies show that uPA is preferentially found in the II/III and V cortical laminae of the gyrencephalic cortex of the non-human primate. Furthermore, we found that in murine cerebral cortical neurons and induced pluripotent stem cell (iPSC)-derived neurons prepared from healthy human donors, most of this uPA is associated with pre-synaptic vesicles. Our in vivo experiments revealed that in both, the gyrencephalic cortex of the non-human primate and the lissecephalic murine brain, cerebral ischemia decreases the number of intact synaptic contacts and the expression of uPA and NCAD in a band of tissue surrounding the necrotic core. Additionally, our in vitro data show that uPA induces the synthesis of NCAD in cerebral cortical neurons, and in line with these observations, intravenous treatment with recombinant uPA three hours after the onset of cerebral ischemia induces NCAD-mediated repair of synaptic contacts in the area surrounding the necrotic core.

Network analysis of the progranulin-deficient mouse brain proteome reveals pathogenic mechanisms shared in human frontotemporal dementia caused by GRN mutations.

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.

The Effects of Physical Activity and Diet Interventions on Body Mass Index in Latin American Children and Adolescents: A Systematic Review and Meta-Analysis.

The objective of this systematic review and meta-analysis is to compare the effect of physical activity only with that of physical activity plus diet interventions on body mass index (BMI) in Latin American children and adolescents. We searched the Medline, Embase, Scopus, Web of Science, and Scielo databases from their inception until March 2020, including studies examining the effect of physical activity or physical activity plus diet interventions on BMI in children and adolescents and based on data from intervention studies. The DerSimonian and Laird method was used to compute a pooled standardized mean difference for BMI in terms of effect size (ES) and respective 95% confidence intervals (CIs). Eighteen studies were included. Analyses were performed based on intervention (four studies were included for physical activity only and four studies were included for physical activity plus diet). In the analysis of physical activity only versus control, there was no effect on BMI (ES = 0.00; 95% CI -0.17-0.17, I2 = 0.0%; p = 0.443). In the analysis of physical activity plus diet versus control, there was a decrease in BMI in favour of the intervention group (ES = -0.28; 95% CI -0.42--0.14, I2 = 74.5%; p = 0.001). When ES was estimated considering only the effect in intervention groups, there was no evidence of a decrease in BMI (ES = -0.17; 95% CI -0.44-0.11, I2 = 84.5%; p < 0.001) for physical activity only (eight studies). However, there was a statistically significant decrease in BMI (ES = -0.30; 95% CI -0.50-0.11, I2 = 95.8%; p < 0.001) for physical activity plus diet (ten studies). Some limitations of this review could compromise our results, but the main limitation that should be stated is the quality of the studies (mainly medium/moderate), especially as physical activity and diet interventions cannot be blinded, compromising the quality of these studies. In summary, this meta-analysis offers evidence that physical activity plus diet interventions produced a reduction in BMI in Latin American children and adolescents, but physical activity only interventions did not.

Urokinase-Type Plasminogen Activator Protects Cerebral Cortical Neurons from Soluble Aß-Induced Synaptic Damage.

Soluble amyloid ß (Aß)-induced synaptic dysfunction is an early event in the pathogenesis of Alzheimer's disease (AD) that precedes the deposition of insoluble Aß and correlates with the development of cognitive deficits better than the number of plaques. The mammalian plasminogen activation (PA) system catalyzes the generation of plasmin via two activators: tissue-type (tPA) and urokinase-type (uPA). A dysfunctional tPA-plasmin system causes defective proteolytic degradation of Aß plaques in advanced stages of AD. In contrast, it is unknown whether uPA and its receptor (uPAR) contribute to the pathogenesis of this disease. Neuronal cadherin (NCAD) plays a pivotal role in the formation of synapses and dendritic branches, and Aß decreases its expression in cerebral cortical neurons. Here we show that neuronal uPA protects the synapse from the harmful effects of soluble Aß. However, Aß-induced inactivation of the eukaryotic initiation factor 2α halts the transcription of uPA mRNA, leaving unopposed the deleterious effects of Aß on the synapse. In line with these observations, the synaptic abundance of uPA, but not uPAR, is decreased in the frontal cortex of AD patients and 5xFAD mice, and in cerebral cortical neurons incubated with soluble Aß. We found that uPA treatment increases the synaptic expression of NCAD by a uPAR-mediated plasmin-independent mechanism, and that uPA-induced formation of NCAD dimers protects the synapse from the harmful effects of soluble Aß oligomers. These data indicate that Aß-induced decrease in the synaptic abundance of uPA contributes to the development of synaptic damage in the early stages of AD.SIGNIFICANCE STATEMENT Soluble amyloid ß (Aß)-induced synaptic dysfunction is an early event in the pathogenesis of cognitive deficits in Alzheimer's disease (AD). We found that neuronal urokinase-type (uPA) protects the synapse from the deleterious effects of soluble Aß. However, Aß-induced inactivation of the eukaryotic initiation factor 2α decreases the synaptic abundance of uPA, leaving unopposed the harmful effects of Aß on the synapse. In line with these observations, the synaptic expression of uPA is decreased in the frontal cortex of AD brains and 5xFAD mice, and uPA treatment abrogates the deleterious effects of Aß on the synapse. These results unveil a novel mechanism of Aß-induced synaptic dysfunction in AD patients, and indicate that recombinant uPA is a potential therapeutic strategy to protect the synapse before the development of irreversible brain damage.

Reference Values for Fitness Level and Gross Motor Skills of 4-6-Year-Old Chilean Children.

In childhood, fitness level is considered an important indicator of health, while gross motor skills are the basis of future motor competence. So far, no reference values have been found for the Chilean population. Therefore, this study aims to provide fitness level and gross motor skill reference values by gender and age of Chilean children aged 4‒6 years. A cross-sectional analysis was conducted that included 728 children between 4 and 6 years old from the La Araucanía region of Chile. To assess the fitness level, the 20-m shuttle run test, standing long jump, handgrip dynamometry, 4x10m shuttle run, and Sit and Reach tests were used. Gross motor skills were assessed by five tests including aiming and catching and balance motor tasks. For fitness level, boys have better values in the long jump and dynamometry test, while girls have better values in flexibility. For estimated maximum volume of oxygen, at 5 years old there are significant differences in favour of boys, while at 6 years old in favour of girls. No statistically significant differences in speed/agility by gender were found. For gross motor skills, boys obtain higher values for catching and aiming tests, and girls for balance. The reference values for fitness level and gross motor skills shown in this study could aid physical education and health professionals in identifying children with low reference values, as well as in establishing objectives that will help to improve their health.

Urokinase-type plasminogen activator (uPA) regulates the expression and function of growth-associated protein 43 (GAP-43) in the synapse.

Growth-associated protein 43 (GAP-43) plays a central role in the formation of presynaptic terminals, synaptic plasticity, and axonal growth and regeneration. During development, GAP-43 is found in axonal extensions of most neurons. In contrast, in the mature brain, its expression is restricted to a few presynaptic terminals and scattered axonal growth cones. Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin and activates signaling pathways that promote cell migration, proliferation, and survival. In the developing brain, uPA induces neuritogenesis and neuronal migration. In contrast, the expression and function of uPA in the mature brain are poorly understood. However, recent evidence reveals that different forms of injury induce release of uPA and expression of uPAR in neurons and that uPA/uPAR binding triggers axonal growth and synapse formation. Here we show that binding of uPA to uPAR induces not only the mobilization of GAP-43 from the axonal shaft to the presynaptic terminal but also its activation in the axonal bouton by PKC-induced calcium-dependent phosphorylation at Ser-41 (pGAP-43). We found that this effect requires open presynaptic N-methyl-d-aspartate receptors but not plasmin generation. Furthermore, our work reveals that, following its activation by uPA/uPAR binding, pGAP-43 colocalizes with presynaptic vesicles and triggers their mobilization to the synaptic release site. Together, these data reveal a novel role of uPA as an activator of the synaptic vesicle cycle in cerebral cortical neurons via its ability to induce presynaptic recruitment and activation of GAP-43.

Switches in histone modifications epigenetically control vitamin D3-dependent transcriptional upregulation of the CYP24A1 gene in osteoblastic cells.

In bone cells vitamin D dependent regulation of gene expression principally occurs through modulation of gene transcription. Binding of the active vitamin D metabolite, 1,25-dihydroxy vitamin D3 (1,25(OH)2 D3 ) to the vitamin D receptor (VDR) induces conformational changes in its C-terminal domain enabling competency for interaction with physiologically relevant coactivators, including SRC-1. Consequently, regulatory complexes can be assembled that support intrinsic enzymatic activities with competency to posttranslationally modify chromatin histones at target genomic sequences to epigenetically alter transcription. Here we examine specific transitions in representation and/or enrichment of epigenetic histone marks during 1,25(OH)2 D3 mediated upregulation of CYP24A1 gene expression in osteoblastic cells. This gene encodes the 24-hydroxylase enzyme, essential for biological control of vitamin D levels. We demonstrate that as the CYP24A1 gene promoter remains transcriptionally silent, there is enrichment of H4R3me2s together with its "writing" enzyme PRMT5 and decreased abundance of the istone H3 and H4 acetylation, H3R17me2a, and H4R3me2a marks as well as of their corresponding "writers." Exposure of osteoblastic cells to 1,25(OH)2 D3 stimulates the recruitment of a VDR/SRC-1 containing complex to the CYP24A1 promoter to mediate increased H3/H4 acetylation. VDR/SRC-1 binding occurs concomitant with the release of PRMT5 and the recruitment of the arginine methyltransferases CARM1 and PRMT1 to catalyze the deposition of the H3R17me2a and H4R3me2a marks, respectively. Our results indicate that these dynamic transitions of histone marks at the CYP24A1 promoter, provide a "chromatin context" that is transcriptionally competent for activation of the CYP24A1 gene in osteoblastic cells in response to 1,25(OH)2 D3 .

Tissue-type plasminogen activator regulates p35-mediated Cdk5 activation in the postsynaptic terminal.

Neuronal depolarization induces the synaptic release of tissue-type plasminogen activator (tPA). Cyclin-dependent kinase-5 (Cdk5) is a member of the family of cyclin-dependent kinases that regulates cell migration and synaptic function in postmitotic neurons. Cdk5 is activated by its binding to p35 (also known as Cdk5r1), a membrane-anchored protein that is rapidly degraded by the proteasome. Here, we show that tPA prevents the degradation of p35 in the synapse by a plasminogen-dependent mechanism that requires open synaptic N-methyl-D-aspartate (NMDA) receptors. We show that tPA treatment increases the abundance of p35 and its binding to Cdk5 in the postsynaptic density (PSD). Furthermore, our data indicate that tPA-induced p35-mediated Cdk5 activation does not induce cell death, but instead prevents NMDA-induced ubiquitylation of postsynaptic density protein-95 (PSD-95; also known as Dlg4) and the removal of GluR1 (also known as Gria1)-containing α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors from the PSD. These results show that the interaction between tPA and synaptic NMDA receptors regulates the expression of AMPA receptor subunits in the PSD via p35-mediated Cdk5 activation. This is a novel role for tPA as a regulator of Cdk5 activation in cerebral cortical neurons.

Urokinase-type plasminogen activator (uPA) protects the tripartite synapse in the ischemic brain via ezrin-mediated formation of peripheral astrocytic processes.

Cerebral ischemia has a harmful effect on the synapse associated with neurological impairment. The "tripartite synapse" is assembled by the pre- and postsynaptic terminals, embraced by astrocytic elongations known as peripheral astrocytic processes (PAPs). Ischemic stroke induces the detachment of PAPs from the synapse, leading to synaptic dysfunction and neuronal death. Ezrin is a membrane-associated protein, required for the formation of PAPs, that links the cell surface to the actin cytoskeleton. Urokinase-type plasminogen activator (uPA) is a serine proteinase that upon binding to its receptor (uPAR) promotes neurite growth during development. In the adult brain, neurons release uPA and astrocytes recruit uPAR to the plasma membrane during the recovery phase from an ischemic stroke, and uPA/uPAR binding promotes functional improvement following an ischemic injury. We found that uPA induces the synthesis of ezrin in astrocytes, with the subsequent formation of PAPs that enter in direct contact with the synapse. Furthermore, either the release of neuronal uPA or intravenous treatment with recombinant uPA (ruPA) induces the formation of PAPs in the ischemic brain, and the interaction of these PAPs with the pre- and postsynaptic terminals protects the integrity of the "tripartite synapse" from the harmful effects of the ischemic injury.

Urokinase-type Plasminogen Activator Induces Neurorepair in the Ischemic Brain.

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. Recent studies indicate that neurons but not astrocytes release uPA during the recovery phase from an ischemic injury, and that binding of uPA to uPAR promotes neurorepair in the ischemic brain by a mechanism that does not require plasmin generation. A combined approach of in vitro and in vivo studies has shown that uPA binding to uPAR induces the reorganization of the actin cytoskeleton in dendritic spines and axons that have suffered an ischemic injury. Furthermore, recent data indicate that uPA-uPAR binding induces astrocytic activation and a crosstalk between activated astrocytes and the injured neuron that triggers a sequence of biochemical events that promote the repair of synapses injured by the ischemic insult. The translational relevance of these observations is noteworthy because following its intravenous administrations recombinant uPA (ruPA) reaches the ischemic tissue, thus raising the question of whether treatment with ruPA is an effective therapeutic strategy to promote neurorepair functional recovery among ischemic stroke survivors.