RESEARCH ARTICLEGenotype and Injury Effect on the Expression of a Novel Hypothalamic Protein Sushi Repeat-Containing Protein X-Linked 2 (SRPX2)
Introduction
According to an estimate, each year 69 million individuals suffer traumatic brain injury (TBI) worldwide (Dewan et al., 2018). TBI leads to tissue damage through cerebral contusion, hemorrhage, altered metabolism and differential gene expression, underlying the evolution of neurologic and psychiatric co-morbidities (Maas et al., 2008). Furthermore, TBI accounts for 15–20% of acquired epilepsy and 5% of all epilepsies (Herman, 2002), leading to development of chronic co-morbidities, resulting in increased disease and economic burden (Pitkänen et al., 2014a). The lack of pharmacotherapies available to improve the post-TBI outcome is a major unmet medical need. Recent data indicate that post-injury remodeling of the extracellular matrix (ECM) is critical for fostering the tissue recovery and alleviation of evolution of co-morbidities, and is thus a potentially rich source for identification of novel treatment targets (Soleman et al., 2013, Pitkänen et al., 2014b).
Urokinase-type plasminogen activator receptor (uPAR) is an essential component of the ECM fibrinolytic system (Sumi et al., 1992, Preissner et al., 2000, Semina et al., 2016). Together with other components of “uPAR-interactome”, consisting of uPAR ligands, urokinase-type plasminogen activator (uPA) and sushi repeat-containing protein X-linked 2 (SRPX2), as well as plasminogen activator inhibitors (PAI-1, PAI-2) (Eden et al., 2011), uPAR-interactome modulates physiological processes like cellular adhesion, proliferation, differentiation and migration through proteolysis in normal and pathologic processes (Sumi et al., 1992, Preissner et al., 2000, Semina et al., 2016). For example, uPAR has been shown to regulate brain plasticity during development and after brain insults (Lahtinen et al., 2006, Liu et al., 2010, Smith and Marshall, 2010, Archinti et al., 2011, Quirico-Santos et al., 2013). Deficiency in uPAR encoding gene, Plaur, results in abnormal development of cortical and hippocampal inhibitory circuitries in Plaur−/− mice (Powell et al., 2003). Furthermore, Plaur deficiency results in more severe epilepsy phenotype and is associated with a delayed inflammatory response in mice after intrahippocampal kainate injection (Ndode-Ekane and Pitkänen, 2013). Deficiency in uPA results in impaired motor recovery and tissue repair after TBI (Lahtinen et al., 2006, Lahtinen et al., 2009, Bolkvadze et al., 2015). Taken together, uPAR interactome plays an important role in post-injury outcome. However, the underlying molecular interactions are not well characterized.
SRPX2 is a novel ligand of uPAR and has been associated with language development, synaptic plasticity, tissue remodeling and angiogenesis during development (Roll et al., 2006, Roll et al., 2010, Royer-Zemmour et al., 2008, Sia et al., 2013). Similar to uPAR, SRPX2 has been associated with epileptogenic alterations in the brain. For instance, recent studies have associated two pathological mutations in the SRPX2 gene in humans with Rolandic epilepsy, oral and speech dyspraxia, mental retardation, and polymicrogyra (Roll et al., 2006, Roll et al., 2010) [but see (Reinthaler et al., 2014)].Co-immunoprecipitation assays demonstrated interaction between SRPX2 and uPAR, and SRPX2 was shown to interact with extracellular domains (DI-III) of uPAR in vitro (Royer-Zemmour et al., 2008). The SRPX2–uPAR interaction initiates neuronal migration and results in cortical excitability (Salmi et al., 2013). In cancer cells, SRPX2 interacts with uPAR on the cell surface and activates P12k/Akt, Ras/MAPK, and P-FAK/Akt pathways, leading to activation of matrix metalloproteinases (MMP2 and MMP9) (Yamada et al., 2014, Tang et al., 2016, Lin et al., 2017, Liu et al., 2017). Therefore, the interaction of SRPX2 with uPAR and their overlapping physiological functions make SRPX2 a promising target for tissue repair and remodeling after acquired brain injury. However, very little is known about the role of uPAR interactome or acquired factors in the regulation of SRPX2 expression.
We recently showed a phylogenetically conserved expression of SRPX2 protein in the paraventricular, periventricular and supraoptic nuclei of the hypothalamus (Anwer et al., 2018). Impaired hypothalamic function has been reported in humans and experimental models after TBI (Colicos et al., 1996, Taylor et al., 2008, Szmydynger-Chodobska et al., 2011, Osterstock et al., 2014, Tanriverdi et al., 2015). For instance, post-TBI reduction in vasopressin levels in patients results in diabetes insipidus indicating damage to the hypothalamo-hypophyseal axis (Capatina et al., 2015). Controlled cortical impact injury (CCI) model of TBI induces a long-lasting growth hormone (GH) deficiency in mice (Osterstock et al., 2014). Additionally, dystrophic neurons were observed in hypothalamic tissue of rats after CCI-induced TBI (Colicos et al., 1996). However, it is not known whether TBI affects hypothalamic SRPX2 expression.
As a first step to understand the factors that influence SRPX2 expression, we hypothesized that a genetic deficiency in uPAR-interactome and/or TBI-induced secondary hypothalamic damage regulates SRPX2 expression in mice. Therefore, we investigated whether SRPX2 expression is altered by a deficiency of its receptor, uPAR and its other ligand, uPA, and to identify TBI-induced changes in SRPX2 expression in wild type and uPAR or uPA-deficient mice. Our data show that SRPX2 expression in the hypothalamus is resistant to the effects of genetic deficiency or acquired injury, whereas uPAR and uPA expression is regulated by CCI.
Section snippets
Study design
To investigate the effect of uPAR or uPA deficiency (genotype effect) and acquired brain injury (injury effect) on SRPX2 expression, three cohorts of mice were used. The overall study design and animal numbers per group are summarized in Fig. 1.
Genotype and injury effects on SRPX2 expression
In order to understand the contribution of uPAR interactome in the regulation of SRPX2 expression in brain, we investigated the effect of uPAR or uPA deficiency and acquired brain injury on SRPX2 expression using immunohistochemistry (cohort 1 and 2) and RT-qPCR (cohort 3).
Discussion
We tested the hypothesis that SRPX2 expression is regulated by uPAR interactome. We investigated whether a deficiency in uPAR, or its key ligand uPA, affects the expression of SRPX2 in brain and if this expression changes following CCI in mice. We found that (i) genetic deficiency of uPAR and uPA did not affect the total number of SRPX2 immunoreactive neurons or Srpx2 gene expression in the hypothalamus; (ii) CCI-induced TBI did not regulate the expression of SRPX2 protein in the hypothalamus
Acknowledgments
This work was supported by the Academy of Finland (A.P.), European Union's Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No. 642881 (ECMED; A.P., M.A.). We thank Mr. Jarmo Hartikainen and Mrs. Merja Lukkari for their excellent technical assistance.
Conflict of interest statement
The authors confirm that there are no conflicts of interest.
Author contributions
M.A., T.B., X.N., N.P., and A.P. Designed the research.
M.A., T.B. Performed the research.
M.A., X.N., N.P., A.P. Analyzed the data.
M.A. and A.P. Wrote the paper with input from all authors.
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