Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome

The function of astrocytes intertwines with the extracellular matrix, whose neuron and glial cell‐derived components shape neuronal plasticity. Astrocyte abnormalities have been reported in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We compared human FXS and control astrocytes generated from human induced pluripotent stem cells and we found increased expression of urokinase plasminogen activator (uPA), which modulates degradation of extracellular matrix. Several pathways associated with uPA and its receptor function were activated in FXS astrocytes. Levels of uPA were also increased in conditioned medium collected from FXS hiPSC‐derived astrocyte cultures and correlated inversely with intracellular Ca2+ responses to activation of L‐type voltage‐gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB within the docking site for the phospholipase‐Cγ1 (PLCγ1), indicating effects of uPA on neuronal plasticity. Gene expression changes during neuronal differentiation preceding astrogenesis likely contributed to properties of astrocytes with FXS‐specific alterations that showed specificity by not affecting differentiation of adenosine triphosphate (ATP)‐responsive astrocyte population. To conclude, our studies identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.

population. To conclude, our studies identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.

K E Y W O R D S
astrocyte, fragile X syndrome, neuronal plasticity, urokinase plasminogen activator

| INTRODUCTION
Astrocytes are important in neuronal maturation and synapse function via bidirectional astrocyte-neuron interactions (Durkee & Araque, 2019;Vasile et al., 2017;Verkhratsky et al., 2014). They provide trophic support to neurons and contribute to brain homeostasis by regulating the extracellular environment. Astrocytes monitor and modulate synaptic transmission by controlling extracellular ion and neurotransmitter concentration in response to cytoplasmic Ca 2+ fluctuations (Südhof, 2018). Molecules produced and released from astrocytes and neurons form extracellular matrix structures, which play important role in cell migration, neurite outgrowth, synaptogenesis, and synaptic plasticity (Song & Dityatev, 2018).
Malfunction of astrocytes is associated with many brain disorders (Molofsky et al., 2012). Deficits of astrocytes are shown to contribute to impaired neuronal function in the brain of Fmr1 knockout (KO) mice, a mouse model of fragile X syndrome (FXS) (Simhal et al., 2019). The syndrome is the most common cause of inherited intellectual disability and a well-characterized monogenic form of autism spectrum disorder (ASD), with a prevalence around one in 4000 males and one in 8000 females (Crawford et al., 1999). The FXS phenotype includes hyperactivity, attention deficits, sensory integration problems, communication difficulties, poor motor coordination, social anxiety, and stereotyped patterns of behavior (Lozano et al., 2016;Terraciano et al., 2005). Epilepsy manifests in around 20% of cases (Berry-Kravis, 2002;Louhivuori et al., 2009) and criteria for ASD are fulfilled in 30%-54% of FXS males (Hagerman et al., 2010). FXS results from the absence of the FMR1 protein (FMRP) caused by transcriptional silencing of the FMR1 gene with the expansion mutation comprising >200 cytosine-guanine-guanine (CGG) trinucleotide repeats (Colak et al., 2014). FMRP is an RNA-binding protein that is essential for normal synapse growth and function (Jin & Warren, 2000).
Coculturing Fmr1 KO neurons with wild type astrocytes rescues the abnormal dendritic phenotype (Jacobs & Doering, 2010), supporting the role for astrocytes in the pathophysiology of FXS.
Selective absence of astroglial FMRP augments neuronal excitability, increases spine density in the motor cortex, and alters the mouse behavioral phenotype (Higashimori et al., 2013;Hodges et al., 2017).
The plasminogen system is affected in the brain of Fmr1 KO mouse appearing increased expression of tissue plasminogen activator (tPA) in neural progenitors and astroglia (Achuta et al., 2014). Fmr1 KO astrocytes express less thrombospondin-1 (TSP-1) (Cheng et al., 2016) that is a matricellular protein, which can mediate synaptic recovery induced by urokinase-type plasminogen activator receptor (uPAR)activated astrocytes (Diaz et al., 2017). Both uPAR and its ligand urokinase (uPA) are highly abundant in the developing central nervous system, and are activated in many disease states (Blasi & Carmeliet, 2002). Upon binding to uPAR, uPA catalyzes plasminogen activation, and plasmin generation as the initiation of the proteolysis cascade, but it also activates cell-signaling pathways involved in the regulation of differentiation, cellular adhesion, migration, and proliferation through non-plasminogenic mechanisms (Blasi & Carmeliet, 2002). Despite potential contribution of uPA/uPAR signaling to the extracellular matrix-linked pathophysiology in FXS (Wen et al., 2018), the signaling system has not been studied with respect to FMRP.  (Achuta et al., 2017;Achuta et al., 2018;Asikainen et al., 2015;Holmqvist et al., 2016;Trokovic et al., 2015). To avoid effects of genetic background, astrocytes were also differentiated from isogenic human embryonic stem cell (hESC) lines : control (H1) and FMR1KO carrying a fragile X mutation that was generated by  (Sayols, 2020). In addition, comparison of the three D95 FXS samples to the two isogenic control samples was performed to detect genes whose final expression significantly differed. Follow-up analyses were performed with the clusterProfiler R package and included pathway enrichment analysis using the REACTOME database (Jassal et al., 2020) to assess significance by hypergeometric test and gene set enrichment analysis (GSEA) ranking genes by their statistics and using gene permutation of the ranked list for calculating the p-value of the enrichment scores.

| Astrocyte conditioned medium
For collection of astrocyte conditioned medium (ACM) cells were plated on Matrigel-coated T25 flasks at a density of 20,000 cells/cm 2 and grown for 7 days. On the day prior to the sample collection, medium was replaced with 5 ml of fresh neurosphere medium. As indicated cells were treated with all-trans retinoic acid (ATRA) for 24 h. Collected medium was filtered through a 0.22 μm filter and stored at À80 C until use.

| Analysis of plasminogen activators in ACM
The quantitative measurements of uPA and tPA in ACM were performed according to the manufacturer's instructions using Abcam's Enzyme-Linked Immunosorbent Assay (ELISA, Abcam) for human uPA (ab119611) and tPA (ab190812). The concentrations in diluted (1:1) culture medium were measured in duplicate, interpolated from the standard curve, and corrected for sample dilution.

| Protein isolation and Western blot analysis
Cells were isolated, suspended in Â1 RIPA lyses buffer (Upstate) supplemented with 1% protease inhibitor cocktail (Sigma), triturated, and centrifuged at Â10,000 g for 10 min at 4 C. Total protein (40 μg) was separated on SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane using Trans-Blot Turbo System (BioRad) and probed with rabbit polyclonal anti-FMRP antibody (1:500, ab17722; Abcam) at 4 C overnight after blocking with 5% nonfat dry milk in PBS at RT for 1 h. After washes, incubation with horseradish-peroxidase conjugated secondary antibody (1:10000, NA9340; GE Healthcare) was performed and immunoreactive protein was visualized using Pierce enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific).

| Calcium imaging
For calcium imaging, astrocytes were plated on coverslips in a 12-well plate with 50,000 cells per well and imaging was performed on the following day as previously described (Achuta et al., 2018). 3 | RESULTS

| Differentiation of astrocytes modeling FXS
We used hPSC-derived forebrain astrocyte model (hASTRO) meaning astrocytes at D95 of differentiation in vitro (Peteri et al., 2020) to study putative alterations of human FXS astrocytes (Figure 1a). Differentiation of astrocytes followed the developmental processes of astrocytes from neuroepithelial induction and regional patterning using morphogens to progenitor expansion and astrocyte specification/maturation (Russ et al., 2021). We monitored the differentiation of isogenic FXS and con- Immunostaining of hASTRO generated from the isogenic FXS and control cell lines and also from four patient-specific FXS and four control hiPSC lines demonstrated that over 90% of cells showed immunoreactivity for established astrocyte markers SOX9, NF1A, GFAP, and S100β (Figure 2a,b). Co-expression of GFAP and S100β was found in 98% of cells, suggesting that the hASTRO represented immature rather than mature astrocytes in the same way as the previously publi-  (Campbell et al., 2006) and MET signaling has been shown to be disrupted in ASD (Campbell et al., 2008). MET is a receptor tyrosine kinase that directly modulates WNT/β-catenin signaling (Kim et al., 2013)  paralogs, SERPINE2 was expressed in FXS and control hASTRO in the same way, while SERPINE3 expression was not detectable in astrocytes as reported previously (Lindner et al., 1986).

| Increased uPA mRNA expression in FXS astrocytes
We used hASTRO differentiated from four FXS male-derived hiPSC lines and three control cell lines to study alterations of plasminogen system in human FXS astrocytes in detail. The expression of PLAU

| Increase in extracellular uPA in FXS astrocyte cultures
To examine secretion of uPA from human astrocytes, we assessed uPA protein levels in ACM collected from control and FXS hASTRO cultures. We found that uPA was increased 5.5-fold (p = .018) in ACM of FXS hASTRO (Figure 5e), suggesting that FXS hASTRO secreted more uPA. As a control, we treated astrocytes with retinoic acid that increases uPA expression (Suzuki et al., 1999); treatment of hASTRO with ATRA increased uPA protein in both FXS and control ACM (Figure 5e). Since our previous studies have shown increased expression of tPA in neural progenitors and brain of Fmr1 KO mice (Achuta et al., 2014), we also measured extracellular levels of tPA that differs in structure from uPA but mediates similar plasminogenic and non-plasminogenic effects in the brain.
Levels of tPA were almost undetectable in ACM (less than 300 pg/ml) and tPA amounts did not differ in FXS and control samples (data not shown).

| Increased extracellular urokinase modulates neuronal plasticity
To elucidate consequences of increased uPA expression in FXS hASTRO, we investigated potential modulatory effects of increased extracellular uPA on neuronal function and thereby possible promaturational effects upon developing networks. Previous studies have shown that binding of neuronal uPA to astrocytic uPAR can activate astrocytes and promote neurological recovery after a hypoxic insult in vivo (Yepes, 2020). To test the neuronal effects of extracellular uPA, we exposed cultured rat primary cortical neurons to ACM col-  (Achuta et al., 2018;Achuta et al., 2017;Boland et al., 2017;Louhivuori et al., 2011;Telias et al., 2015;Utami et al., 2020). The uPA-mediated astrocytic mechanisms could serve as critical regulatory Data are means ± SEM. Two-way ANOVA and Fisher's LSD, ***p < 0.001 mechanisms maintaining cellular homeostasis that is compromised in the absence of FMRP, but they may also provide cellular feedback loop linked to neuronal BDNF/TrkB signaling (Louhivuori et al., 2011), which when dysregulated, could contribute to impaired neuronal plasticity and the phenotype of FXS.
The absence of FMRP altered the expression of several genes leading to FXS-specific changes during sequential differentiation of neural progenitors to functional astrocytes. However, the number of differentially expressed genes in FXS hASTRO at D95 of differentiation was limited to 81 genes when compared to controls. Abnormalities in the plasminogen system, especially related to uPA/uPAR emerged clearly in FXS hASTRO. Our data suggest that astrocytic uPA/uPAR signaling could provide an essential regulatory mechanism that convey messages from extracellular space to define functional properties of both astrocytes and neurons. Upon binding to its receptor, uPA catalyzes the conversion of plasminogen to plasmin on the cell surface, but uPAR also is able to engage in multiple protein/ protein interactions (Blasi & Carmeliet, 2002). Since uPAR is devoid of a transmembrane and cytoplasmic domain, it needs a transmembrane partner to activate intracellular signaling. Integrins, G-protein receptors, and caveolin are mediators of uPA/uPAR signaling (Blasi & Carmeliet, 2002). We observed that increased uPA/uPAR signaling in playing roles in dendrite, and axon development (Oliva et al., 2018).
Given that activation of WNT/β-catenin signaling can repress astrocyte specification (Sun et al., 2019), augmented WNT signaling together with reduced functional responses through L-type VGCCs in FXS hASTRO may indicate that lack FMRP affects specification astrocytes.
Both uPA and uPAR are developmentally regulated and their expression declines after peaking at the postnatal period to low or undetectable levels in the normal adult brain (Del Bigio et al., 1999). A transient increase of astrocytic uPAR is seen after cerebral ischemia (Yepes, 2020). In addition, involvement of the plasminogen system and uPA/uPAR signaling has been reported in brain tumors, multiple sclerosis, Alzheimer's disease, epilepsy, and developmental brain malformations (Akenami et al., 1997;Baart et al., 2020;Gveric et al., 2001;Iyer et al., 2010;Liu et al., 2010). Furthermore, a genetic link between the PLAUR gene and autism has been established integrated with MET signaling (Campbell et al., 2008). Involvement of uPAR in ASD was indicated by increased PLAUR mRNA expression in the postmortem ASD brain. The present study connected alterations of uPA/uPAR signaling with FXS, the most common genetic cause of ASD. The regulatory role of astrocytes in uPA/uPAR signaling and correlations of uPA levels with functional properties of astrocytes have not been previously described and our data provide novel insights to basic cellular mechanisms that could be disrupted in several forms of ASD. Impact of uPAR signaling in the developmental processes that define the adaptive capacity of cortical circuits was previously demonstrated in transgenic mice with genetic deletion of uPAR resulting in a reduced number of parvalbumin interneurons (Eagleson et al., 2010).
Thus, uPA/uPAR signaling could contribute to synaptic excitation and inhibition balance that is affected in FXS and in many neurodevelopmental disorders (Gibson et al., 2008). Increased uPA/uPAR signaling in FXS hASTRO may reflect the abnormality and inability of astrocytes lacking FMRP to provide the normal neuronal support needed. Indeed, previous studies have demonstrated that coculturing of Fmr1 KO mouse neurons with wild type mouse astrocytes shows rescue effects on the neuronal phenotype caused by FMRP deficiency (Jacobs & Doering, 2010).
Astrocytes comprise morphologically and functionally diverse populations of cells with brain region-specific differences in their properties. The differentiation method we employed produced homogenous cultures of human forebrain astrocytes expressing canonical astrocytic markers. The hASTRO were functional, as demonstrated by using intracellular calcium recordings, and allowed investigation of the innate properties of FXS astrocytes.
Astrocytes are crucial for synaptic and neuronal network formation and function, maintenance of brain homeostasis, and response to injury and repair. The astrocyte secretome illustrates astrocytic function. Rat primary cortical neurons were well suited for screening of the neuronal effects of FXS astrocyte secretome. Using a standardized method, murine primary neuronal cultures can be produced with minimal contribution of nonneuronal cells in a reliable and reproducible manner (Sahu et al., 2019). Since neurons do not divide in culture and they can be maintained for a limited period in culture, primary cortical, and hippocampal cultures need to be generated from embryonic or early postnal brain regions every time. Neuronal differentiation from stem cells, an unlimited source of progenitors, is current option that also allows generation of human neurons. Functional neurons can be differentiated from hiPSCs or fibroblast-direct conversion, but there are some variation in the differentiation efficiency depending on the used differentiation protocol. Effects of FXS hASTRO secretome remain to be validated in hiPSC-derived human neuronal cultures.
Altered functional maturation of FXS hASTRO and effects of FXS hASTRO secretome on TrkB signaling in rat neurons suggested that astrocytes have a significant impact on neuronal network formation and function in FXS. Therefore, understanding of the special role of astrocytes in the FXS brain is important for the success of treatment strategies.

ACKNOWLEDGMENTS
We would like to thank the DNA Sequencing and Genomics Laboratory at the Institute of Biotechnology for the RNA sequencing analysis and Pia Laine together with Petri Auvinen for assisting with the data analysis. Imaging experiments were performed using services of the Biomedicum Imaging Unit. We also thank the Director of the Neuroscience Center Jari Koistinaho for the control hiPS cell lines. The work was supported by the Academy of Finland, the Arvo and Lea Ylppö