Ventricular enlargement associated with the panneural ablation of the podocalyxin gene

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Abstract

Podocalyxin (Podxl) is a type I membrane mucin-protein of the CD34 family abundantly expressed in kidney epithelial cells (podocytes) where it plays a crucial functional role. Podxl is also expressed in tissues other than kidney, like in brain, but its function is ignored. To investigate the functional role of podocalyxin (Podxl) in brain we produced the specific brain-ablation of the Podxl gen in mice by crossing Podxlfloxed/floxed mice, generated in our laboratory, to mice with pan-neural expression of recombinase Cre (Cre3).

Podxl−/− mice show no apparent behavioral phenotype but their brains showed enlargement of ventricular volumes detected in vivo by MR imaging. The pattern of brain vasculature was of normal appearance but the thickness of the main carotid artery was significantly increased. Moreover, the histological analysis showed increased number of choroidal capillaries lining the ventricular spaces. These findings are analyzed in the light of the role likely played by podocalyxin in cell migration and cell–cell recognition during brain development and also on the consistent findings of increased ventricular spaces in human pathological disorders like schizophrenia.

Introduction

Podocalyxin (PODXL), also known as Myb-Ets transformed progenitor (MEP), is a mucin protein of the CD34 family originally described in the epithelial intraglomerular cells of kidney lining the capillary surfaces (podocytes) (Kerjaschki et al., 1984). The strong negative charge of PODXL is the reason for its affinity for cationic stains (Seiler et al., 1977), reason why it was initially described as the kidney epithelial polyanion. The estimated mass of the PODXL peptide is 59 kDa that undergoes extensive posttranslational processing to yield a mature native protein of ∼ 165 kDa (Kershaw et al., 1997). PODXL is a type I membrane protein with a mucin-like amino terminus domain, negatively charged due to heavy sialylation and sulfation.

Besides the epithelial kidney glomeruli, PODXL is also expressed in megakaryocytes and platelets (Miettinen et al., 1999, McNagny et al., 1997), vascular endothelial cells, brain, liver and heart (Kershaw et al., 1995, Horvat et al., 1986, Testa et al., 2009). It is also found in hematopoietic cell precursors (McNagny et al., 1997, Hara et al., 1999, Kerosuo et al., 2004, Doyonnas et al., 2005), many human tumors (Schopperle et al., 2003, Somasiri et al., 2004, Casey et al., 2006) and also in embryonal carcinoma stem cell (Bhattacharya et al. 2004). PODXL is essential for the renal function since null Podxl−/− mice die of anuria immediately after birth (Doyonnas et al., 2001). PODXL was postulated to exert the anti-adhesive force necessary to maintain open the podocyte interdigitations, “filtration membrane slits” (Kerjaschki et al., 1984, Takeda et al., 2000). In contrast, PODXL adheres to endovascular l-selectin in high endothelial venules (Somasiri et al., 2004) and cells expressing recombinant podocalyxin showed enhanced adherence, migration and cellular intercommunication (Larrucea et al., 2007, Larrucea et al., 2008). The latter effects are mediated by P-selectin and integrins and showed dependency on Ca2+ calmodulin and sialylation (Larrucea et al., 2007, Larrucea et al., 2008). Moreover, the metastatic ability of tumor cells seems to correlate with the degree of podocalyxin expression.

Expression of PODXL has also been reported in brain, showing highest levels in the cortical plate, hippocampus, cerebellum and basal forebrain nuclei (Vitureira et al., 2005, Lin et al., 2007). In humans, PODXL has been associated with the malignant progression of brain astrocytic tumors (Hayatsua et al., 2008) and it has also been considered a candidate gene responsible for the hereditary congenital facial paresis (van der Zwaaga et al., 2005). The function of PODXL in brain is ignored. Thus, we found of interest to analyze the eventual structural/functional changes associated with the brain-specific ablation of the Podxl gene (brain-Podxl−/−).

Magnetic resonance imaging (MRI) and spectroscopy (MRS) methods provide non-invasive and comprehensive information of cerebral morphology and metabolism in normal and genetically engineered mice (Budinger et al., 1999, Strome and Doudet, 2007). While MRI is able to provide quantitative information on cerebral morphology including the vascular network, MRS provides detailed information on the cerebral energetic state. In this work, we combined the use of MRI and MRS approaches with classical immunological staining methods to characterize, in vivo and in vitro, the cerebral morphology and metabolic pattern of normal, and brain-Podxl-deficient mice. MRS analysis showed no detectable changes in the pattern of observable cerebral metabolites, but NMR imaging demonstrated a consistent increase in the ventricular volume as well as in the size of the brain vessels.

Section snippets

Podocalyxin expression in brain

This work aimed at investigating whether the absence of Podxl in brain would induce any specific developmental and/or functional perturbation. We first tested whether crossing Podxl floxed mice (Podxlfloxed/floxed) to mice expressing the Cre transgene specifically in brain (Cre3) would effectively remove the brain Podxl. The presence of brain Podxl was estimated by Western blotting of total brain homogenates (Fig. 1). The Podxl content of null brain-Podxl mice showed a decrease of 77%. Since

Discussion

Despite numerous pieces of information, the precise functional role of the sialoprotein Podxl in the nervous system remains to be elucidated. To determine the functional role of Podxl, Doyonnas et al. (2001) studied the structural features of brains from Podxl knock out mice. However, since the lack of Podxl in the kidney results in anuria and death immediately after birth, the usefulness of this experimental model is limited. Nevertheless, newborn mice with systemic ablation of the Podxl gene

Animals

The animals were maintained under controlled temperature and light cycles with free access to chow and water. The animal Care Committee of the Center for Biological Research (CSIC) approved all the experimental procedures performed on the mice. Cre3 mice with pan-neural expression of recombinase Cre were obtained from Dr. Krajewski (Banares et al., 2005). Mice with panendothelial expression of recombinase Cre (Tie2) were obtained from Dr. Kisanuki (Kisanuki et al., 2001).

Ablation of podocalyxin gene (Podxl) in the central nervous system

We produced

Acknowledgments

Adam Nowakowski holds a research predoctoral contract from de CIBERER. Susana Larrucea present address: Research Unit, Hospital de Cruces, Baracaldo, Vizcaya, 48903-Spain. Sonia Alonso-Martín was recipient of a predoctoral fellowship from the Basque Community (BF101-40). Authors are indebted to the valuable suggestions and critical reading of the manuscript and to the SIERMAC for providing expert technical assistance in the NMR imaging and spectroscopy experiments. This work was supported in

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